Producing blood cells from stem cells could yield a purer, safer cell therapy

The journal Stem Cells Translational Medicine has published a new protocol for reprogramming induced pluripotent stem cells (iPSCs) into mature blood cells. This protocol uses only a small amount of the patient’s own blood and a readily available cell type. This novel method skips the generally accepted process of mixing iPSCs with either mouse or human stromal cells. Therefore, is ensures that no outside viruses or exogenous DNA contaminates the reprogrammed cells. Such a protocol could lead to a purer, safer therapeutic grade of stem cells for use in regenerative medicine.

The potential for the field of regenerative medicine has been greatly advanced by the discovery of iPSCs. These cells allow for the production of patient-specific iPSCs from the individual for potential autologous treatment, or treatment that uses the patient’s own cells. Such a strategy avoids the possibility of rejection and numerous other harmful side effects.

CD34+ cells are found in bone marrow and are involved with the production of new red and white blood cells. However, collecting enough CD34+ cells from a patient to produce enough blood for therapeutic purposes usually requires a large volume of blood from the patient. However, a new study outlined But scientists found a way around this, as outlined by Yuet Wai Kan, M.D., FRS, and Lin Ye, Ph.D. from the Department of Medicine and Institute for Human Genetic, University of California-San Francisco has devised a way around this impasse.

“We used Sendai viral vectors to generate iPSCs efficiently from adult mobilized CD34+ and peripheral blood mononuclear cells (MNCs),” Dr. Kan explained. “Sendai virus is an RNA virus that carries no risk of altering the host genome, so is considered an efficient solution for generating safe iPSC.”

“Just 2 milliliters of blood yielded iPS cells from which hematopoietic stem and progenitor cells could be generated. These cells could contain up to 40 percent CD34+ cells, of which approximately 25 percent were the type of precursors that could be differentiated into mature blood cells. These interesting findings reveal a protocol for the generation iPSCs using a readily available cell type,” Dr. Ye added. “We also found that MNCs can be efficiently reprogrammed into iPSCs as readily as CD34+ cells. Furthermore, these MNCs derived iPSCs can be terminally differentiated into mature blood cells.”

“This method, which uses only a small blood sample, may represent an option for generating iPSCs that maintains their genomic integrity,” said Anthony Atala, MD, Editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “The fact that these cells were differentiated into mature blood cells suggests their use in blood diseases.”

Mesenchymal Stem Cells Rarely Engraft But Work in a “Hit and Run” Manner

Even though this paper was published in 2012, it is a very important study that deserves a wide reading and lots of discussion.

The paper is “Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long-Term Engraftment and No Ectopic Tissue Formation” from Kathleen Le Blanc’ s laboratory, which was published in Stem Cells 2012;30:1575–1578.

In this paper, Le Blanc and her colleagues examined autopsies from patients who had received mesenchymal stem cell transplants. Since many scientists consider mesenchymal stromal cells (MSCs) a novel treatment for a variety of medical conditions, it is crucial that the fate of MSCs after infusion is better understood. Also, the long-term safety profile of MSCs is also quite important. DO they cause malignant transformation and ectopic tissue formation? Autopsies are an excellent way to address this questions.

The Le Blanc laboratory examined autopsy material from 18 patients who had received MSC transplants from people other than themselves. They analyzed 108 tissue samples from 15 patients by means of polymerase chain reaction (PCR) to search for the DNA of MSCs from donors in the tissue. If such foreign DNA was present in the tissues of the stem cell recipients, this would indicate that the MSCs had engrafted into the tissues of the patient. Unfortunately, MSC donor DNA was detected in only one or several tissues including lungs, lymph nodes, and intestine in eight patients at very low levels (from 1/100 to <1/1,000). Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, which simply means that the more time had elapsed since the time of the MSc transplant, the less likely it was that MSC DNA was found in the patient. For example, MSC DNA was detected in nine of 13 patients whose MSC infusions had been given within 50 days before sampling, in only two of eight of those infusions that had been given earlier.

On a more positive note, there were no signs of ectopic tissue formation or malignant tumors of MSC-donor origin upon macroscopic or histological examination of the tissues of the autopsied individuals.

What does all this mean? MSCs appear to mediate their healing capacities through the molecules that they secrete. This is called a “paracrine” mechanism. and MSCs seem to engraft into host tissues only very rarely. Instead MSCs come to a damaged tissue and stimulate the endogenous healing mechanisms already present. After doing this job, MSCs do not typically stick around. Thus, MSCs seem to work in, what Le Blanc calls a “hit and run” mechanism.

Because MSCs do not seem to engraft over a long period of time, the potential adverse reactions to these cells seems to be largely limited. Thus these cells are quite safe, but their effects are almost certainly indirect to some extent.

Mesenchymal Stem Cell Article

I wrote this review article for the Mesenchymal Stem Cell site.  Unfortunately, this site has now become defunct.  Therefore, I have moved it here for your enjoyment:

“Critical Distinctions between Mesenchymal Stem Cells from Bone Marrow and Alternative Sources”

Michael Buratovich Ph.D (Author)
Supplied Courtesy of BioInformant Worldwide, LLC

Mesenchymal stem cells (MSCs) are adult, multipotent stem cells that have been isolated from circulating blood (Kuznetsov et al 2001), umbilical cord blood (Beibacket al 2004; Lee et al 2004b), placenta (Iguraet al 2004), heart (Warejckaet al 1996), amniotic fluid (Tsai et al2004), adipose tissue (Katzet al 2005), synovium (Fickert et al 2003), skeletal muscle (Younget al 1995), pancreas (Hu et al 2003), deciduous teeth (Estrelaet al 2011), and bone marrow (Charbord 2010). Bone marrow-derived MSCs (BMSCs) are the most heavily-studied of all MSCs, and, therefore, tend to be the standard against which MSCs from other sources are evaluated. BMSCs can differentiate into osteoblasts, chondrocytes, adipocytes, fibroblasts, hepatocytes, neural cells, etc., and can give rise to cartilage (Kadiyala et al 1997), bone (Bruder et al 1997; 1998), tendon (Young et al 1998), muscle (Galmiche et al 1993; Ferrari et al 1998), and many other tissues. Do MSCs from tissues other than bone marrow have similar differentiation potentials, and if not how does the potency of these MSCs from alternative sources compare with those from bone marrow? Fortunately stem-cell scientists have examined this question in some detail, but a central question remains: Do MSCs from diverse bodily locations represent distinct or the same cell types?

If MSCs throughout the body are similar cell types then we would expect them to have similar embryological origins. However, this is not the case, since MSCs develop from several different embryonic tissues. The first wave of MSCs arises from Sox-1-expressing neuroepithelial cells during embryonic development. However, later MSCs come from multiple sources (Takashima et al 2007), including neural crest cells (Nagoshi et al 2008; Morikawaet al 2009). Therefore, MSCs from various tissues almost certainly have distinct embryological origins. Additionally, MSCs are located in different sites in the body, and are influenced by specific microenvironments. Thus MSCs from different tissue sources might represent distinct cell types, and could potentially display distinct differentiation profiles and express particular genes. Despite these differences in developmental origin and environmental influences, MSCs from various sources have very similar morphologies and share a common array of surface markers (Mitchell et al 2003; Lee et al 2004a; Wang et al 2004; Tsai et al 2007). However, several studies have established that MSC populations are rather heterogeneous (Dominici et al 2009), and, therefore, surface markers expressed on some cells of an MSC population are not always expressed in all the cells of that population (Mafi et al 2011). Also, the growth kinetics of cultured MSCs differs remarkably with respect to their source (Kang et al 2004b; Yoshimura et al 2007; Troyer and Weiss 2008).

Despite the shared array of cell surface markers, presently there are no cellular markers or cell surface proteins that are unique to MSCs. In order to provide a more unified approach to MSC biology, the International Society of Cryotherapy has proposed three criteria for the identification of MSCs. Under these criteria, MSCs must: (1) be plastic-adherent when maintained in standard culture conditions; (2) express the following cell surface molecules CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules, and; (3) be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro (Dominici et al 2006). Despite these definitions, flow cytometric analyses of MSCs from several different populations have shown some significant differences in cell surface markers (Boeuf and Richter 2010). For example, even though the absence of CD34 is generally considered a criterion for the definition of MSCs, various investigators have reported low expression of CD34 in ADSCs (ADSCs; De Ugarte et al 2003a; Rebelatto et al 2008; Roche et al 2009) and BMSCs (Zvaifler et al 2000; Gronthos et al 2003; Yu et al 2010). Likewise, many investigators have shown that MSCs from multiple sources do not express CD45 (Zvaifler et al 2000; Zuk et al 2002; Igura et al 2004; Dominici et al 2006; Wongchuensoontorn et al 2009), but BMSCs are CD45 positive (Yu et al 2010).

Other cell marker differences include CD271,which shows high levels of expression in BMSCs and ADSCs (Jones et al 2002; Quirici et al 2010), but is not expressed in synovial membrane MSCs (SMSCs; De Bari et al 2001; Van Landuyt et al 2010). Another molecule that is highly expressed in the vast majority of MSC population is STRO-1 (Gronthos et al 1991; Simmons and Torok-Storb 1991; Gronthos et al 1994; Gronthos et al 1999; Stewart et al 1999; Walsh et al 2000; Zuk et al 2002; Miura et al 2003; Kadar et al 2009), but other studies have shown that ADSCs are STRO-1 negative (Gronthos et al 2001). Signal transduction receptors also show varied expression in distinct MSC populations. For example, platelet-derived growth factor receptor (CD140a/PDGFRα) is involved in proliferation and migration of osteoblasts and MSCs. This receptor is much more highly expressed in SMSCs than BMSCs (Nimura et al 2008). Finally the vascular cell adhesion molecule CD106/VCAM1, which is involved in hematopoietic stem cell homing (Simmons et al 1992), is more highly expressed in BMSCs than ADSCs (De Ugarte et al 2003a; Kern et al 2006; Rider et al 2008; Roche et al 2009). This cell surface difference almost certainly is related to the specific microenvironment in which BMSCs are found and their specific roles in maintaining hematopoietic stem cell growth.

Comparative gene array analyses of MSCs from different sources have revealed some differences in gene expression between these distinct MSC populations, but overall the gene expression profiles between these cells are relatively similar (Winter et al 2003; Lee et al 2004a; Djouad et al 2005; Wagner et al 2005; Aranda et al 2009; Jansen et al 2010). Proteomic comparisons of distinct MSC populations using two-dimensional gel electrophoresis analysis came to very similar conclusions (Roche et al 2009). MSCs from intra-articular tissues (synovial membrane and anterior cruciate ligament) and chondrocytes show gene expression profiles that were more similar to each other than to MSCs from extra-articular locations (Segawa et al 2009). These data suggest that MSCs from varied sources probably represent similar, but distinct cell types that express a core of common genes, but also clusters of distinct genes. These gene expression differences convey different differentiation potentials upon specific MSC populations and varied requirements for these particular MSC populations to differentiate into specific cell types (Gimble et al 2008; Rastegar et al 2010).

MSC Differentiation
With respect to the differentiation potential of MSC populations, the general rule of thumb is the closer the MSC source tissue is to the target tissue, the more effectively that particular MSC population differentiates into the target tissue. A few examples should suffice. Yoshimura and colleagues found that rat SMSCs derived from the synovial tissue of the knee, which is closest to the target tissue of chondral cartilage, formed cartilage better than BMSCs, ADSCs, or MSCs from periosteum or muscle (Yoshimura et al 2007). Likewise, gene expression profiles of human BMSCs or umbilical cord-derived MSCs (UCSCs from Wharton’s jelly) definitively showed that BMSCs express a variety of osteogenic genes (RUNX2, DLX5 and NPR3) not observed in UCSCs. Under osteogenic induction, BMSCs produced far more bone than UCSCs. However, UCSCs express angiogenesis genesand fewer genes involved in the immune response than BMSCs, suggesting that UCSCs are superior for allogeneic transplantation. When cocultured with allogeneic macrophages,UCSCs prevented the macrophages from producing immunomodulatory cytokines tumor necrosis factor and Interleukin-6 (Hsieh et al 2010). Finally, Niemeyer and coworkers showed that BMSCs and ADSCs formed bone with similar efficiencies in vivo (Niemeyer et al 2007), but in animals studies, BMSCs produced better repair of tibial osteochondral defects in sheep when compared to ADSCs (Niemeyeret al 2010).

MSC Chondrogenesis
Initiation of cartilage development during animal development begins with the condensation of mesenchymal precursor cells (Woods, Wang and Beier 2007). These cell-cell contacts are mediated by N-cadherin, whose expression is highly upregulated in human MSCs after being subjected to chondrogenic induction (Tuli et al 2003). N-cadherin is required for chondrogenesis of chick limb mesenchymal cells in vitro and in vivo (Oberlender and Tuan 1994). Prior to MSC condensation prechondrocytic MSCs secrete extracellular matrix rich in hyaluronic acid, collagen type I and IIa. Initiation of MSC condensation also correlates with the expression of neural cadherin (N-cadherin) and neural cell adhesion molecule (N-CAM). The secreted signaling molecule transforming growth factor-β (TGF-β) is one of the earliest signals in chondrogenic condensation. TGF-β activates production of the extracellular matrix protein fibronectin, which up-regulates N-CAM, and also stimulates the synthesis of Sox transcription factors (Sox-5, -6 and -9), which are essential for cartilage formation. Other extracellular matrix molecules made by chondrogenic MSCs include tenascins, thrombospondins, and cartilage oligomeric protein (COMP). These extracellular matrix molecules interact with cell adhesion molecules to activate intracellular signaling pathways that initiate the transition from chondroprogenitor cells to fully committed chondrocytes. Proliferating chondroprogenitor cells synthesize hyaluronan, collagen II, IX and XI, and the cartilage-specific proteoglycan core protein (or chondroitin sulfate proteoglycan 1) known as aggrecan. Aggrecan (encoded by the ACANgene) is a member of the aggrecan/versican proteoglycan family, and is the most predominant proteoglycan in the extracellular matrix of articular cartilage. Aggrecan helps cartilage withstand compression. N-cadherin and N-CAM expression fade and disappear in differentiating chondrocytes (Golding, Tsuchimochi and Ijiri 2006).

When grown under chondrogenic conditions, MSCs in monolayer culture respond by condensing into high-density three-dimensional cell aggregates (Winter et al 2003). In order to realistically recapitulate chondrogenesis in culture, researchers deposit centrifuged MSC pellets that contain ~200,000 – 500,000 cells in a two-dimensional culture. This culture system, which is one of the most widely used in chondrogenesis research, is called a pellet, aggregate or spheroid culture. To induce chondrogenesis, pellets are cultured in a basal medium (typically low- or high-glucose Dulbecco’s Modified Eagles Medium, otherwise known as DMEM, or fetal calf serum) that contains dexamethasone, ascorbate, proline, insulin, transferrin and selenous acid (Johnstone et al 1998; MacKay et al 1998; Puetzer, Petitte and Loboa 2010). Classically, the growth factor used to induce chondrogenesis in this type of medium is 10 ng/ml of transforming growth factor-β (TGF-β). TGF-β1, 2, and 3 are the only well-established full inducers of chondrogenesis that, when added as single factors, induce proteoglycan and collagen type II deposition (MacKay et al 1998; Barry et al 2001). Other chondrogenic inducers have been described; bone morphogen protein-2 (BMP-2) for BMSCs (Schmitt et al 2003) and BMP-6 for ADSCs (Estes, Wu and Guilak 2006). However, other studies have failed to confirm the chondrogenic efficacy of these two growth factors (Winter et al 2003; Indrawattana et al 2004; Xu et al 2006; Hennig et al 2007; Weiss et al 2010), and there is even a chance that these two growth factors might only work in a donor-specific fashion.BMP-2, -4, and -6, and insulin-like growth factor-1 (IGF-1) seem to promote chondrogenesis in MSCs when given in combination with TGF-β (Schmitt et al 2003; Im, Shin and Lee 2005; Sekiya et al 2005; Liu et al 2007).

Presently, a significant controversy exists over whether ADSCs or BMSCs are better sources for orthopedic tissue repair (Frisbee et al 2009). Both BMSCs and ADSCs have been successfully differentiated into chondrocytes in vitro (John stone et al 1998; Erickson et al 2002) and used for cartilage repair in vivo (Wakitani et al 1994; Im et al 2001; Centeno et al 2011). However, harvesting adipose tissue is much less painful than bone marrow aspirations, which makes ADSCs much more preferable for orthopedic therapies.

With respect to MSC chondrogenesis (cartilage induction), several studies have reported relatively robust chondrogenesis by ADSCs in two-dimensional (Zuk et al 2002; Erickson et al 2002; Gimble and Guilak 2003) and three-dimensional culture systems (Awad et al 2004; Estes, Wu, and Guilak 2006). However, several head-to-head comparisons of BMSCs and ADSCs have produced contradictory results, with some studies reporting equivalent chondrogenic capacities (Zuk et al 2001; De Ugarte et al 2003b; Rebelatto et al 2007), but many others concluding that human and equine BMSCs show superior chondrogenic ability (Winter et al 2003; Im, Shin and Lee 2005; Sakaguchi et al 2005; Vidal et al 2008). Because the same MSC populations from different donors show different differentiation potentials (Bieback et al 2004; Chang et al 2006a Kern et al 2006), head-to-head comparisons of donor-matched MSC populations are essential in order to compare the chondrogenic potential of MSCs that share the same genetic background. Such donor-matched studies have consistently shown that BMSCs show superior chondrogenic potential over ADSCs (Huang et al 2005; Afizah et al 2007). Additionally, gene array studies indicate that during chondrogenic induction, BMSCs show gene expression profiles that more closely resemble native cartilage than ADSCs (Winter et al 2003). If grown in three-dimensional culture, which is thought to be an essential aspect of chondrogenic differentiation (Johnstone et al 1998; Yoo et al 1998; Erickson et al 2002), once again BMSCs outperform ADSCs if seeded in a hyaluronic acid scaffold (Jakobsen et al 2010) or encapsulated in alginate (Mehlhorn et al 2006). BMSCs also show superior chondrogenesis to UCSCs in a three-dimensional culture in which cells were seeded in a polygycolic acid (PGA) matrix (Wang et al 2009).

These data do not necessarily mean that BMSCs are the best cartilage-making MSCs in the body. First of all, head-to-head comparisons treated both MSC populations with the same chondrogenic induction protocol, which implicitly assumes that culture conditions optimized for BMSCs are also be optimal for ADSCs. This assumption, however, ignores the intrinsic differences between these two MSC populations. Kim and Im have shown that ADSCs display a chondrogenic potential equal to that of BMSCs if ADSCs are treated with higher concentrations of growth factors (Kim and Im 2009). Additionally, Diekman and colleagues have shown that chondrogenesis of BMSCs and ADSCs is highly dependent on the presence and concentration of particular growth factors, the presence or absence of serum, and the composition of the scaffold in which the cells are embedded for the chondrogenic induction. ADSCs made significantly more aggrecan in response to BMP-6 than to TGF-β, but the opposite was true for BMSCs. Likewise, ADSCs produced more type II collagen in the presence of serum whereas BMSCs produced more type II collagen without serum. Finally when seeded in alginate beads, the quantity of glycosaminoglycan (GAG) made by BMSCs were significantly higher in the dual-growth factor cocktail of TGF-β and BMP-6 as compared to TGF-β alone. However, when these same cells were grown in a cartilage-derived matrix, those grown in the TGF-β-alone cocktail had higher viability and produced higher amounts of GAG when compared to those grown in dual cocktail (TGF-β + BMP-6). Thus the growth scaffold greatly influences the response of MSCs to particular growth factors, but these data also underscore that BMSCs and ADSCs are probably distinct cell types (Diekman et al 2010).

Secondly, keeping with the original rule that the closer the source tissue is to the desired target tissue, the more effectively MSCs from those tissue sources differentiate into the target tissue, Sakaguchi and colleagues showed that MSCS from bone marrow, synovium, and periosteum made more cartilage than ADSCs or skeletal muscle-derived MSCs, but SMSCs clearly made the most cartilage (Sakaguchi et al 2005). Interestingly, this result was replicated in rat MSCs (Yoshimura et al 2007). Equine BMSCs, however, do show superior chondrogenesis to UCSCs and MSCs from amniotic fluid (Lovati et al 2011), and human fetal and adult BMSCs exceed the chondrogenic potentials of fetal lung-, and placenta-derived MSCs (Bernardo et al 2007).

The varied responses of MSCs from various sources to different growth factors also have been well documented. For example, TGF-β alone is sufficient for chondrogenesis of BMSCs (Afizah et al 2007), but not ADSCs (Awad et al 2003: Estes, Wu and Guilak 2005). Additionally, the combination of TGF-β and dexamethasone stimulates chondrogenesis in BMSCs, but in ADSCs, TGFβ is required for chondrogenesis but dexamethasone tends to suppress chondrogenesis (Awad et al 2003). The reduced chondrogenic induction of ADSCs by TGF-β is probably due to reduced expression of the TGF-β receptor in these cells. However, BMP-6 treatment induces expression of the TGF-β receptor ALK-5 in ADSCs and combined application of TGF-β and BMP-6 restores chondrogenesis in this MSC population (Hennig et al 2007). A published protocol to successfully differentiate ADSCs into chondrocytes makes use of the combination of TGF-β and BMP-6 (Estes et al 2010).

Differential responses to BMP-6 are also observed in different types of MSCs. As previously mentioned, BMP-6 strongly induces chondrogenesis in ADSCs, but not in BMSCs. BMP-6 in combination with TGF-β inhibits hypertrophy in ADSCs (Estes, Wu and Guilak 2003), but in BMSCs, BMP-6 promotes hypertrophy and endochondral ossification (Sekiya, Colter and Prockop 2001; Sekiya et al 2002; Indrawattana et al 2004).

These varied responses to growth factors by distinct MSC populations might also be a reflection of the assorted levels of “stemness” found among the cells of each MSC population. As previously noted, MSC populations tend to be highly heterotropic, and clonal analyses of ADSCs have shown that these cell populations are a mixture of cells that can form bone, cartilage and fat (tripotent), those that can only form two of these tissues (bipotent), and others that can only form only cell type (monopotent). The ratios of these tripotent, bipotent to monopotent clones seems to vary from study to study. Guilak and colleagues found that 21% of ADSCs clones were tripotent and approximately 30% were bipotent (Guilak et al 2006), but Zuk and others found that only 1.4% of all ADSC clones were tripotent (Zuk et al 2002). The disparities between these studies seem to be due to the media conditions used, the age of the adipose tissue donors, and the overall design of the experiment. However, these studies certainly show that distinct MSC populations consist of cells at varying levels of “stemness,” with some being more committed to a particular cell type and others being less developmentally committed to a particular cell fate. The heterogeneity of these populations almost certainly influences the response of these cell populations to particular growth factors.

MSC Osteogenesis
Runt-related transcription factor-2 (Runx-2) is considered a master regulator of early osteogenic differentiation (Fujita et al 2004). In combination with TGF-β, Runx-2 up-regulates the expression of interleukin-11 (IL-11), which reduces adipogenesis (fat formation) and promotes chondrocytic and osteocytic differentiation (Enomoto et al 2004). Runx-2 also promotes the expression of osterix, another important osteogenic inducer. Osterix suppresses chondrogenesis at low concentrations and promotes osteogenesis at high concentrations (Tominaga et al 2009).

Continuous exposure of BMSCs or ADSCs to ligands for the glucocorticoid receptor (e.g., dexamethasone)and/or the vitamin D receptor (e.g., 1,25 dihydroxyvitamin D3), plus ascorbic acid and β-glycerophosphate induces them to produce mineralized extracellular matrix within three weeks (Gimble et al 2008).Exposure of MSCs to BMPs and Wnt signaling proteins also results in successful differentiation into osteoblasts (Peng et al 2003; Shea et al 2003; Kang et al 2004a; Luo et al 2004; Peng et al 2004; Si et al 2006; Luu et al 2007; Deng et al 2008; Tang et al 2009). Additionally, magnetic field stimulation and can also stimulate osteogenic differentiation of MSCs (Singh, YashRoy and Hoque 2006).

Several studies have found that ADSCs and BMSCs from humans and other animals show equal osteogenic potential (Zuk et al 2001; Zuk et al 2002; De Ugarte et al 2003b; Winter et al 2003; Cowan et al 2004; Lee et al 2004a; Romanov et al 2005; Wagner et al 2005; Kern et al 2006). However, other studies argue that BMSCs display superior osteogenic potential to ADSCs (Im, Shin and Lee 2005; Sakaguchi et al 2005; Musina et al 2006; Lui et al 2007; Yoshimura et al 2007). Yet another study insists that ADSCs have superior osteogenic potential than BMSCs (Izadpanah et al 2006).

In head-to-head comparisons with other types of MSCs, the osteogenic potential of BMSCs was approximately the same as SMSCs, and only slightly better than periosteum-derived MSCs (Sakaguchi et al 2005). However, in another study SMSCs from healthy donors expressed significantly lower levels of osteogenic markers after induction of osteogenesis (Djouad et al 2005). Another comparison between human umbilical cord perivascular cells (HUCPVCs) and BMSCs found that HUCPVCs had higher osteogenic potential than BMSCs (Baksh, Yao and Tuan 2007). However, other studies compared the gene expression profiles and osteogenic potential of UCSCs and BMSCs not only showed a pronounced expression of osteogenic genes in BMSCs, but also established their superior osteogenic potential in in vitro differentiation assays (Hsieh et al 2010; Majore et al 2011). It is unclear if these two experiments analyzed the same umbilical cord cell populations. MSCs isolated from human umbilical cord blood also showed a distinctly greater osteogenic potential in comparison to BMSCs (Chang et al 2006a). Also human UCSCs show superior osteogenic potential in comparison to chorionic plate-derived MSCs (Kim et al 2011).

MSC Adipogenesis
Adipocytes are specialized cells that store triacylglycerols (fats). MSC differentiation into adipocytes requires the activity of a transcription factor called peroxisome proliferator activator receptor-gamma (PPAR-γ). PPAR-γ regulates the function of many adipocyte specific genes (Rosen 2000), and interacts with members of the CCAAT/enhancer binding protein (C/EBP) family to regulate adipogenesis (Farmer 2005). Osteogenic transcription factor Runx2 inhibits adipogenesis by directly interacting with PPAR-γ (Akune et al 2004).

Adipogenic induction of cultured MSCs requires the use of compounds that increase intracellular levels of the signaling molecule 3’,5’-cyclic adenosine monophosphate (cAMP) such as phosphodiesterase inhibitors (e.g., isobutylmethylxanthine or theophylline), and ligands for the glucocorticoid receptor (e.g., dexamethasone), and PPAR-γ, (i.e., rosiglitazone, which is marketed as the anti-diabetic insulin sensitizer AvandiaTM). Additionally, most adipogenic cocktails also include insulin, and some protocols also include indomethacine (Mosna, Sensebe and Krampera 2010). MSCs exposed to these agents form intracellular droplets composed of neutral lipid and express key adipogenic markers (e.g., adiponectin, fatty acid binding protein, aP2) within three-to-nine days (Gimble et al 2008; Muruganandan, Roman and Sinal 2009).

Head-to-head comparisons of MSCs from varied tissue sources have shown that ADSCs have an adipogenic potential that is superior (Sakaguchi et al 2005; Izadpanah et al 2006; Musina et al 2006; Liu et al 2007; Yoshimura et al 2007; Rider et al 2008) or equal to that of BMSCs (Zuk et al 2001; 2002; De Ugarte et al 2003b; Winter et al 2003; Lee et al 2004a; Romanov et al 2005;Wagner et al 2005; Kern et al 2006). SMSCs also showed an adipogenic potential that was equal to that of ADSCs and superior to that of periosteum-derived MSCs (Sakaguchi et al 2005; Yoshimura et al 2007). Some studies suggest that UCSCs show poor adipogenic ability in comparison to BMSCs and ADSCs (Rebelatto et al 2008; Hsieh et al 2010), but another study found that HUCPVCs had superior adipogenic potential when compared to BMSCs (Bask, Tao and Tuan 2007). Chorionic-plate-derived MSCs showed superior adipogenic potential to UCSCs (Kim et al 2011), but umbilical cord and umbilical cord blood seem to contain more than one MSC population, all of which display different adipogenic potentials (Chang et al 2006b; Kestendjieva et al 2008; Cheong et al 2010; Lu et al 2010; Majore et al 2011).

MSC Muscle Differentiation
Myogenesis (muscle formation) is regulated by a family of transcription factors known as the myogenic regulatory factors (MRFs). During embryonic development, two basic helix-loop-helix (bHLH) transcription factors, MyoD and Myf5, establish the skeletal muscle lineage and drive myocyte differentiation (Rudnicki et al 1993). Later events in myogenesis that consist of myocyte fusion into myotubes and the synthesis of muscle-specific contractile proteins is associated with the expression of another bHLH transcription factor, myogenin (Hasty et al 1993; Nabeshima et al 1993). Muscle injury activates a muscle stem cell population called satellite cells that recapitulate the MRF expression program (Smith et al 1994; Yablonka-Reuveni and Rivera 1994; Cornelison and Wold 1997; Cooper et al 1999).

Many different types of MSCs can form skeletal, smooth and cardiac muscle. Maintaining MSCs in 10%-20% serum causes them to express smooth muscle markers like α-smooth muscle actin (Abedin, Tintut and Demer 2004; Gimble et al 2008). When transplanted in vitro, MSCs make smooth muscle rather easily (Galmiche et al 1993; Wakitani, Saito and Caplan 1995; Prockop et al 1997; Ferrari et al 1998; Pittenger et al 1999; Caplan and Bruder 2001; Jiang et al 2002).

Exposing MSCs to low serum concentrations or horse serum leads to the expression of skeletal muscle markers such as myogenin and the formation of multi-nuclear myotubes. However, MSCs do not differentiate into mature, skeletal muscles as readily as they do smooth muscles, and the culture conditions under which the cells are grown seem to be extremely important. Co-culturing BMSCs (Lee, Kosinski and Kemp 2005; Beier et al 2011) or ADSCs (Di Rocco et al 2006) with skeletal muscles can induce myotube formation and the expression of myogenic genes by MSCs. The efficiency of skeletal muscle formation with this procedure is almost doubled by exposing MSCs to the chromatin remodeling reagent trichostatin A (Collins-Hooper et al; 2011). Incubation of MSCs with conditioned medium prepared from chemically damaged, but not undamaged, muscle cells also induces MSC myotube formation and expression of MyoD (Santa Maria, Rojas and Minguell; 2004). Treatment of MSCs with particular molecules such as Galectin-1 (Chan et al 2006), TWEAK (Gigenrath et al 2006) and 5-azacytidine (Kocaefe et al 2010; Natasuke et al 2010) can also induce myogenesis, as can hypoxic preconditioning (Leroux et al 2010).

Dezawa and colleagues have published a protocol for differentiating BMSCs into skeletal muscle. They treated mouse BMSCs for three days with a mixture of bFGF, forskolin, which is known to increase intracellular concentrations of cAMP, platelet-derived growth factor and neuregulin. After the three-day culture period, they transfected the cells with a plasmid that encoded the intracellular domain of the Notch receptor, and selected only those cells that had successfully taken up the plasmid. To augment the ability of the remaining cells to form myotubes, they exposed the cells to either 2% horse serum or ITS (insulin-transferrin-selenite) in serum-free medium. Both of these media promoted myogenic differentiation of MSCs to myoblasts that formed myotubes, and were able to integrate into existing muscle and repair muscle in mdx mice (Dezawa et al 2005). mdx Mice harbor a loss-of-function mutation in the gene that encodes the dystrophin protein, which, in humans, is defective in individuals who are afflicted with Duchenne Muscular Dystrophy (Muntoni, Torelli and Ferlini 2003). Therefore, even though it shows a relatively mild phenotype, the mdx mouse is a model system for muscular dystrophy (Sicinski et al 1998).

Treatment of MSCs with a drug called 5-azacytidine directs them to transdifferentiate into cells that resemble cardiomyocytes (heart muscle cells). In cells, 5-azacytidine is incorporated into DNA where it inhibits DNA methylation, and DNA hypomethylation leads to activation of particular genes (Christman 2002). Treatment of BMSCs (Fukuda 2001; Shim et al 2004; Xu et al 2004; Antonitsis et al 2007; 2008), ADSCs (Rangappa et al 2003b; Lee et al 2009) or UCSCs (Cheng et al 2003) with 5-azacytidine drives them to form cells that have a fibroblast-like morphology, synchronously beat, and express many cardiac-specific genes like troponin T, atrial natriuretic protein (ANP), GATA-4, Nkx2.5, TEF-1, and MEF-2C (Fukuda 2001; 2002; Yang et al 2012). Some work has even shown that these differentiated MSCs respond to adrenergic and muscarinic stimulation (Fukuda 2002), and can integrate into the heart of a laboratory animal and form functional connections with native cardiomyocytes (Hattan et al 2005).

MSCs can also be converted into cardiomyocytes by being co-cultured with living (Rangappa et al 2003a; Yoon et al 2005b; Arminan et al 2009; Peran et al 2010) or apoptotic cardiomyocytes (He et al 2010). Also treatment with particular growth factors, such as BMP-2, Fibroblast growth factor -2 (FGF-2) and IGF-1 (Yoon et al 2005a; Bartunek et al 2007; Hahn et al 2008), can push MSCs to become cardiomyocytes, as can transfection with particular genes like Wnt-11 (He et al 2011), GATA-4 (Li et al 2011), or a combination of GATA-4 and Nkx2.5 (Gao, Tan and Wang 2011). Some controversy exists over cardiomyocyte-induced MSCs, since some studies suggest that differentiated MSCs retain their stromal phenotypes and are, at best, only immature cardiomyocytes (Gallo et al 2007; Rose et al 2008).

Because MSC populations tend to form smooth muscle rather readily, there have been few head-to-head comparisons of the efficiency of smooth muscle formation in distinct MSC populations.

Comparisons of the ability of various MSC populations to differentiate into skeletal muscles include in vitro differentiation of MSCs from bone marrow, spleen, thymus, and liver. This study showed that BMSCs, liver- and thymus-derived MSCs all made skeletal muscle in culture, but splenic-derived MSCs did not (Gornostaeva, Rzhaninova and Gol’dstein 2006). Comparisons of the in vivo ability of BMSCs, ADSCs, and SMSCs to form skeletal muscle when implanted showed that ADSCs had the greatest ability to integrate into existing muscles (de la Garza-Rodea et al 2011).

Interestingly, a small fraction of BMSCs can form myotubes and integrate into existing muscle when injected into laboratory animals, whether that muscle is damaged or not (Ferrari et al 1998), a characteristic also shared by SMSCs (De Bari et al 2003). However, when UCSCs were injected into the tail vein of mdx mice, the cells were able to integrate into the muscle but unable to differentiate in vivo into mature, skeletal muscles (Vieira et al 2010; Zucconi et al 2011). Different MSCs show varying efficiencies of cardiomyocyte differentiation. UCSCs, for example, show particularly low transdifferentiation rates (Martin-Rendon et al 2008). ADSCs, however, transdifferentiate into cardiomyocytes with the highest efficiency (Zhu et al 2008; Tobita, Orbay and Mizuno 2011; Paul et al 2011;Yong et al 2012). In fact, when grown in a semisolid methycellulose medium enriched with growth factors, ADSCs spontaneously form beating ventricular- and atrial-like cardiomyocytes (Planat-Benard et al 2004). This makes ADSCs an attractive source of material for cardiac regenerative therapies.

MSCs and Tooth Formation
Tooth formation results from a complex set of interactions between the overlying stomadial epithelium and underlying mesenchymal cells. Dental mesenchymal cells develop from neural crest cells derived from midbrain and hindbrain cranial neural crest cells. In mice, these two cell populations are in place by day 8.5 (E8.5) and by day 10.5 (E10.5) tooth-forming sites and tooth types are determined. At E11.5, a localized thickening of the dental epithelium that results from cell shape changes forms the “dental placode.” Between E12.5-E13.5, the dental placode proliferates and invaginates to form the epithelial bud around which mesenchymal cells condense (Peters and Bailing 1999). At E14.5, the cap stage, the epithelial component of the developing tooth folds and forms a transient cluster of non-dividing cells called the “enamel knot.” The enamel knot is a signaling center that produces many powerful growth factors, including Sonic hedgehog (Shh), BMP-2, BMP-4, BMP-7, FGF-4 and FGF-9 (Thesleff and Mikkola 2002). The cap stage is followed by the bell stage, and at this time the epithelially-derived ameloblasts and the mesenchymally-derived odontoblasts differentiate. The ameloblasts form enamel and the odontoblasts produce the dentine. MSCs also generate the alveolar bone that forms the sockets for the teeth. Human tooth development occurs in a very similar fashion (Zhang et al 2005).

In adult animals, dentinal repair results from odontoblasts that differentiate from a precursor cell population that resides in dental pulp tissue. These dental pulp stem cells (DPSCs) have been isolated from adult human teeth (Gronthos et al 2002). In culture, DPSCs show robust growth and a high proliferation rate and, even after extensive subculturing, have the ability to form a dentin/mineralized complex with a mineralized matrix when grafted into the dorsal surface of immunocompromised mice (Gronthos, et al 2002; Batouli et al 2003). In a rabbit model of tooth regeneration, DPSCs are able to support the formation of functional teeth (Hung et al 2011), and in mouse and dog models, DPSCs regenerated alveolar tooth socket bone in the jaw (Yamada et al 2010; 2011; Ito et al 2011).

Four other dental-associated, MSC-like stem cell populations have been isolated and characterized. The first of these, stem cells from human exfoliated deciduous teeth (SHED), like DPSCs, have many similarities to MSCs. However, SHEDs differ from DPSCs in that they have a higher proliferation rate and can differentiate into odontoblasts, which form a dentin-pulp-like structure without the mineralized matrix, but not ameloblasts (Miura et al 2003). Transplantation experiments have established that SHEDs can make vascularized bone and endothelial cells, and when implanted into the jaws of laboratory animals SHEDs can effectively regenerate jaw bone (Cordeiro et al 2008; Nakamura et al 2009; Yamada et al 2010; 2011; Ito et al 2011). The second cell population, periodontal ligament stem cells (PDLSCs), expresses a subset of neural crest cell and MSC markers (Seo et al 2004; Nagatomo et al 2006; Gay et al 2007; Fujita et al 2007; Coura et al 2008; Huang et al 2009), and shows some ability to repair periodontium (Seo 2004; Grimm et al 2011). The third population, stem cells from apical papillae (SCAP) readily makes dentin-pulp-like complexes and expresses several neuronal markers (Sonoyama et al 2006; 2008). The fourth stem population, dental follicle precursor cells (DFPCs), form fibrous and rigid tissue when transplanted into laboratory animals but not dentin, cementum or bone (Morsczecket al 2005; 2008).

In a head-to-head comparison of the ability of DPSCs and ADSCs to replace teeth in a rabbit model, the teeth produced by ADSCs were very similar to those generated by DPSCs. Both sets of replacement teeth were living teeth with nerves and vascular systems, but the ADSCs grew at faster rate and were more resistant to senescence (Hung et al 2011). BMSCs, like DPSCs, are also able to form calcified deposits in vitro (Gronthos et al 2000). Likewise, gene microarray analyses of these two stem cell populations show similar levels of expression for more than 4000 genes, with only a few differences (Shi, Robey and Gronthos 2001). Head-to-head comparisons of BMSCs, DPSCs, and SHEDs have shown that these stem cells have an equivalent the ability to regenerate alveolar tooth socket bone in the jaws of laboratory animals (Yamada et al 2010; 2011; Ito et al 2011). Comparison of BMSCs and SHED gene expression profiles by means of DNA microarray and real-time reverse transcriptase polymerase chain reaction has shown that 2753 genes in SHEDs show a more than two-fold difference in expression level in comparison to BMSCs. The genes that show the greatest differences in expression in SHEDs are those involved in BMP signaling, and the protein kinase A (PKA), c-Jun-N-terminal kinase (JNK), and apoptosis signaling-regulating kinase-1 (ASK-1) signaling cascades. Therefore SHEDs have specific characteristics that differ from BMSCs, and the osteogenic and odontogenic differentiation of SHEDs and BMSCs are probably regulated by different mechanisms (Hara et al 2009).

BMSCs can probably serve as a source for dental regenerative treatments, but the faster growth rates and easier isolation of ADSCs probably makes them a superior choice.

MSC Neural Differentiation
To date, neural differentiation of MSCs remains controversial, since many stem cell biologists think that the neuron-like cells formed by MSCs after neural induction do not represent true neurons. However, protocols have been published for converting MSCs into specific types of neurons. One method (Tropel et al 2006) cultures MSCs at low density (3,000 cells / cm2) on poly-lysine-coated plates for seven days in low-glucose DMEM, 10% fetal calf serum, glutamine (2mM), and bFGF (25ng/mL). A second protocol incubates MSCs with bFGF (5ng / mL) for 24 hours, followed by complete medium substitution with DMEM, N2 supplement, butylated-hydroxyanisole, KCl, valproic acid, and forskolin (Krampera et al 2007; Anghileri et al 2008). When subjected to either protocol, MSCs show dramatic morphological changes after 24-48 hours. They begin to sprout long branches and axon-like structures. Molecularly, neurally induced MSCs up-regulate synthesis of the neuron-specific intermediate filament nestin, which is typically only made by dividing neurons and disappears from terminally differentiated neurons (Michalczyk and Ziman 2005). Neurally induced MSCs also initiate expression of several neuronal and glial markers that include light neurofilament (NF-L), β-tubulin III (β3-tub), peripheral myelin protein-22 (PMP-22), glial fibrillary acidic protein (GFAP), and NeuN or neuronal nuclear antigen (Krampera et al 2007). They also express functional neuronal receptors and pharmacologically sensitive voltage-gated calcium channels (Wislet-Gendebien et al 2005; Tropel et al 2006). Unfortunately, MSC neuronal induction is reversible, and as soon as neural induction ceases MSCs revert back to their ground state. Interestingly, co-culturing neutrally induced MSCs with Schwann cells locks the neutrally induced MSCs in their neuronal state (Krampera et al 2007).

Despite reports that MSCs can be differentiated into functional neurons, several studies have failed to recapitulate these results (Scuteri et al 2010). Time-lapse photography of rat BMSCs that had undergone neural induction showed that instead of extending neurites, the cells merely shrunk and retracted their cell extensions so that only two extensions remained. This was interpreted to be a response to toxic or stressful conditions, and treatment of MSCs with chemicals and conditions known to stress cells (extremes of pH, high-molarity NaCl or detergents) produced similar “pseudoneuronal” morphology and increased MSC staining for neuronal markers. Strangely, pretreatment of MSCs with cycloheximide (an antibiotic that inhibits translation) failed to abrogate this response, suggesting that no new gene expression is required for cells to assume this pseudoneuronal morphology. These findings suggest that neural induction of MSCs in culture is largely an artifact (Lu, Blesch and Tuszynski 2004). Other studies have implanted MSCs into the brains of laboratory animals in the hope that a neural environment can induce neuronal differentiation in MSCs, but the implanted cells showed a spherical morphology with few extensions and connections with other cells (Zhao et al 2002).

Despite these negative results, genetic engineering of MSCs with the intracellular domain of Notch (Dezawa et al 2004; Xu et al 2010), neurogenin-1 (Kim et al 2008), neurotrophin-3 after retinoic acid pretreatment (Zhang et al 2006), siNRSF (Yang et al 2008) and brain-derived neurotrophic factor (Lim et al 2011), have all successfully transdifferentiated MSCs into functional neurons. Furthermore, MSC treatment with various combinations of growth factors (Long et al 2005; Bae et al 2011; Trzaska and Rameshwar 2011), signaling molecules (Kondo et al 2011) and small molecules (Wang et al 2011) have also transdifferentiated MSCs into neurons, and in some cases into dopaminergic neurons. Finally, sequential analysis of gene expression (SAGE) and microRNA expression profiles of MSCs before and after neural induction have shown high level expression of several neural specific genes that are not expressed in MSCs before neural induction. Also cell the expression of reprogramming factors like Oct4, Klf4, and c-Myc are modulated during differentiation (Crobu et al 2011).

With respect to MSC neuronal differentiation, BMSCs have definitely received the most attention. However, other types of MSCs have the capacity to form neuron-like cells (Chen, He and Zhang 2009; Chang et al 2010; Jiang et al 2010; Lim et al 2010). To date there have been few head-to-head comparisons of the efficiency of neural induction between distinct MSC populations, and this is probably a function of the variability of MSC neural induction. One study found that neural induction of UCSCs and BMSCs produces dopaminergic neurons with roughly equal efficiencies (Datta et al 2011).

Also, there are few comparisons with dentally-derived MSCs, but these cells descend from neural crest cells. Consequently, they demonstrate more neural properties than other types of MSCs (Karaoz et al 2011). Such MSCs begin with more neural characteristics, and, therefore, neural differentiation of dental-derived MSCs probably requires fewer molecular steps (Nourbakhsh et al 2011).

Are BMSCs significantly different or relatively similar to MSCs from other tissue sources? The extensive research on BMSCs has provided a wealth of data that we can use for comparison with other MSCs. Work on MCSs from other tissues strongly suggests that genuine similarities exist between BMSCs and other types of MSCs. All these MSCs, with a few exceptions, display roughly the same set of cell surface proteins (De Ugarte et al 2003a; Musina, Bekchanova and Sukhikh 2005). For the most part, clonal differences in specific MSC populations notwithstanding (Zuk et al 2002; Guilak et al 2006), can differentiate into osteocytes, chondrocytes, or adipocytes (Pittenger et al 1999; Pontos et al 2006), and BMSCs and ADSCs utilize common pathways to differentiate into these distinct cell types (Liu et al 2007). They also express a common core of genes and proteins that distinguish them from other cell types.

Despite these similarities, there are also some stark differences between various MSCs from assorted tissues. First of all, the efficiencies with which these different MSC populations differentiate into osteocytes, chondrocytes, and adipocytes widely differ. Secondly, even though BMSCs and ADSCs use a set of common genes for early differentiation into all three lineages, they recruit different sets of genes for later differentiation and maturation into fully differentiated cells (Liu et al 2007; Kim and Im 2010). Thirdly, varied MSC populations differ with regards to their stemness. UCSCs share more genes in common with embryonic stem cells than BMSCs, and are, therefore, more primitive. They also express more angiogenesis and growth related genes. On the other hand, the gene expression profiles of BMSCs are much more significantly altered under different culture conditions, and express more osteogenesis genes (Hsieh et al 2010). Fourth, even though MSC populations commonly express a core set of genes(Winter et al 2003; Lee et al 2004a; Djouad et al 2005; Wagner et al 2005; Aranda et al 2009; Jansen et al 2010), gene expression profiles of distinct MSC populations differs substantially. For example, UCSCs and umbilical cord blood-derived MSCs(UBSCs) show remarkable differences in gene expression. Gene expression profiles from UBSCs revealed that genes involved in anatomical structure and multicellular organism development, osteogenesis and the immune system were expressed at high levels. However in UCSCs, genes related to cell adhesion, neurogenesis, morphogenesis, secretion and angiogenesis were more highly expressed (Secco et al 2009). Fifth, even though distinct MSC populations express very similar sets of proteins (Roche et al 2009), there are significant differences (Maurer 2011). Finally, the differentiation requirements for each MSC population differ, and these differences are a result of the signature gene expression profiles of each MSC population.

Thus, MSCs represent a familial cell type, but each distinctive MSC population represents a particular subfamily of this cell type family. While some subfamilies are clearly more closely related to some than others, these MSC subfamilies constitute the constituents that compose the MSC cell type.

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Michael Buratovich received his Ph.D. in Cell and Developmental Biology from UC Irvine in the laboratory of Peter Bryant where he worked on tumor suppressor genes in Drosophila melanogaster. He worked as a postdoctoral research fellow at Sussex University with Robert Whittle on the role of Wnt proteins in patterning the peripheral nervous system, and at University of Pennsylvania with Betsy Wilder on Wnt signaling during development. Since 1999, he has been a member of the faculty of Spring Arbor University (Spring Arbor, MI) in the Biochemistry department. He also served as a visiting scientist at Boston University in the laboratory of Joseph Ozer where he worked on the role of basal transcription factors in stem cell differentiation, and has collaborated with Amr Amin at the University of Al-Ain on cancer research. He is zealous about communicating science to the public and passionately blogs at

Stem Cell Treatments for Bladder Dysfunction

Spina bifida is a birth defect in which the backbone and spinal canal do not close before birth. The damage to the central nervous system affects various organs that receive nervous inputs from the spinal cord and one such organ is the urinary bladder. A condition called “neurogenic bladder” results from an inability of the nervous system to properly control the urinary bladder and the muscle tissue that lines the wall of the bladder. Neurogenic bladder can lead to spasms and a pressure build-up in the bladder. This results in urinary incontinence, and children with spina bifida and neurogenic bladder often have an urge to urinate after drinking comparatively small amounts of liquid. They can also involuntarily leak urine, and this creates a great deal of social embarrassment and emotional stress. If untreated, the long-standing and frequent pressure build-up in the bladder can lead to infections and even kidney damage.

Spina bifida

Surgical treatments for neurogenic bladder involve reconstruction of the bladder that increases its size by grafting patches from the patient’s bowel. Because the graft comes from the patient’s own body, it is unlikely that the immune system will reject these grafts. Also, the intestinal tissue patches are, on the average, strong enough to withstand the pressures in the bladder. However, there is a certain incompatibility between intestinal and bladder tissue, and this can cause long-term complications that include urinary tract infections, urinary tract stones and, rarely, cancers. Thus, researchers have been searching for newer safer patches which resemble the actual bladder wall.

Northwestern University researchers have published a study that used stem cells of children with spina bifida to generate tissue patches for bladder surgery. This paper, “Cotransplantation with specific populations of spina bifida bone marrow stem/progenitor cells enhances urinary bladder regeneration,” was published in the Proceedings of the National Academy of Sciences. Arun Sharma and colleagues from Earl Cheng’s laboratory isolated two types of cells from the bone marrow of child spina bifida patients. They isolated mesenchymal stem cells (MSCs) and CD34+ cells, which are stem and progenitor cells that usually give rise to blood cells. Sharma and her colleagues used these cells to coated molds that were made with a special polymer scaffold called POC (poly(octanediol-co-citrate). These cell-filled molds were used to create a patch graft that was transplanted into rat bladders. This type of surgery is not that unlike the bladder augmentation surgery used for spina bifida patients.

Next, the Cheng lab workers determined if the human tissue survived in the implanted patch. In those cases where both human cell types (MSCs and CD34+) were combined, over half the implanted patch was covered with muscle tissue, four weeks after the implantation. However, if only CD34+ cells were used, only a quarter of the patch was covered with muscle tissue. Interestingly, the implanted patch also showed evidence of some peripheral nerve growth and blood vessel formation, both of which are found in healthy, normal bladder walls. These experiments demonstrate suggest that a patient’s own bone marrow stem cells can be used to help construct a tissue patch that can potentially act as a graft patch for bladder augmentation surgeries. Also, since some nerve growth in the implanted patch was observed, and this definitely an exciting result. Could it be possible to re-connect the reconstructed bladder tissue with the main nervous system? Possibly, but the most severe cases of neurogenic bladder are almost certainly more difficult cases to treat successfully.


Despite the exciting possibilities in this study, there are some caveats. First of all, was the muscle formed from the stem cells or the surrounding tissue? It is not clear from this paper, and since implantation of an empty POC scaffold without any human stem cells results in 20% coverage with muscle tissue, at least some of the newly formed muscle tissue is actually derived from the host rat and not from human stem cells. Secondly, how do these patch grafts compare to the intestinal patches? This was not assessed. Finally, rats with neurogenic bladder were not implanted, and this is an important control, since it is at least possible, that the muscle growth would be less robust in an animal with neurogenic bladder. Thus, despite this great potential shown in this paper, several questions remain.

A second paper used a very different approach. Debra Franck and others in Joshua R. Mauney’s lab at Harvard Medical School coated a silk thread scaffold with extracellular matrix proteins and coated with smooth muscle cells that were made from induced pluripotent stem cells into the smooth muscle cells. Unfortunately, Franck and colleagues did not evaluate the newly created patch in a living animal. Also, this paper used mouse induce pluripotent stem cells, and it is not clear that human induced pluripotent stem cells would be able to do the same thing.

While these two studies are strictly experimental, they might provide some new avenues of research for new treatments for neurogenic bladder.

FDA Approves Argus II Retinal Prosthesis

The Food and Drug Administration (FDA) of the United States has approved the first retinal implant for use in the United States. This approval is for Second Sight’s Argus II Retinal Prosthesis System, which provides limited sight to those patients blinded by a rare genetic eye condition called advanced retinitis pigmentosa. This condition damages the light-sensitive cells that line the outer layer of the retina and causes them to die. This severely reduces vision and eventually leads to blindness.

Argus II

Second Sight has devoted more than 20 years of research and development to the development of the Argus II Retinal Prosthesis. It has succeeded in two clinical trials, and the funding for the development of this device – more than $200 million – came from the National Eye Institute, the Department of Energy and the National Science Foundation. The remaining money came from private investors. European regulators approved the Argus II for use in 2011 and it has been used in 30 patients in clinical-trial patients since 2007. The Ophthalmic Devices Advisory Panel of the FDA unanimously recommended approval for the Argus II in September 2012.

The Argus II includes a small video camera, a video processing unit and a 60-electrode implanted retinal prosthesis with a transmitter mounted on a pair of eyeglasses. This device replaces the function of degenerated cells in the retina. It must be stressed that the Argus II does not fully restore vision, but it can improve a patient’s ability to perceive images and movement. It uses the video processing unit to transform images from the video camera into electronic data that is wirelessly transmitted to the retinal prosthesis.

Retinitis pigmentosa affects about one in 4,000 people in the US and about 1.5 million people worldwide. It kills off the retina’s photoreceptors, which convert light into electrical signals that are transmitted by means of the optic nerve to the brain’s visual cortex for processing. Second Sight plans to adapt its technology to assist people afflicted with age-related macular degeneration, which is a similar but more common disease.

Second Sight has plans to make the Argus II available later this year in clinical centers throughout the US. They want to establish a network of surgeons who have the skills to implant the device and, eventually recruit hospitals to offer it.

The Argus II is not the only retinal implant under development. A medical start-up company called Retina Implant AG uses a different approach in its device. In this case, the prosthetic device, the Alpha IMS Implant, is inserted beneath a portion of the retina. The three- by three-millimeter microelectronic chip (0.1-millimeter thick) contains ~1,500 light-sensitive photodiodes, amplifiers and electrodes. The Alpha IMS Implant is surgically inserted beneath a portion of the retina known as the fovea (which contains a rich concentration of particular photoreceptors known as cone cells) in the retina’s macula region. The fovea enables the highest clarity of vision for people to read, watch TV and drive. This chip helps generate at least partial vision by stimulating intact nerve cells in the retina. The nerve impulses from these cells are then fed by means of the optic nerve to the visual cortex where they create impressions of sight. The power source for the chip is implanted under the skin behind the ear and connected by a thin cable to the chip. In May the company announced its first UK patients for its latest trial. To date surgeons have implanted the Alpha IMS Implant prosthetic in 36 patients through two clinical trials over six years.

Alpha IMS Implant

Researchers from Stanford University researchers are developing self-powered retinal implants in which each pixel in the device is fitted with silicon photodiodes. These sensors detect light, and control the output of a pulsed electric current. Patients would be required to wear a set of goggles for these devices that emit near-infrared pulses that transmit power and data directly to the photodiodes. Inductive coils that must be surgically implanted in the patient’s head to power these other retinal prostheses. This design was reported in May 2012 issue of Nature Photonics, and in the article, they described in vitro electrical stimulation of healthy and degenerate rat retina by photodiodes powered by near-infrared light.

Other researchers are utilizing yet another design for retinal prosthesis design. Researchers from Weill Cornell Medical College in New York City have deciphered the neural codes that mouse and monkey retinas use to turn light patterns into patterns of electrical pulses that their brains translate into meaningful images. Next they programmed this information into an “encoder” chip that was combined with a mini-projector to create an implantable prosthetic. This chip converts images that come into the eye into a series of electrical impulses, and the mini-projector then converts the electrical impulses into light impulses that are sent to the brain. With this approach, instead of increasing the number of electrodes placed in an eye to capture more information and send signals to the brain, this approach increases the quality of the artificial signals themselves, which improves their ability to carry impulses to the brain.

Mesenchymal Stem Cell Transplantation Improves Heart Remodeling After a Heart Attack

Stem cell scientists from the University of Maryland, Baltimore have used bone marrow mesenchymal stem cells (MSCs) to treat sheep that had suffered a heart attack. They found that the injected stem cells prevented the heart from deteriorating.

This work was a collaboration between the laboratories of Mark Pittenger, ZhonGjun Wu and Bartley Griffith from the Department of Surgery and the Artificial Organ Laboratory.

After a heart attack, the region of the heart that was deprived of oxygen undergoes cell death and is replaced by a heart scar. However, the region next to the dead cells also undergo problematic changes. The cells in these regions adjacent to dead region must contract more forcibly in order to compensate for the noncontracting dead region. These cells enlarge, but some undergo cell death due to inadequate blood supply. There are other changes that can occur, such as abnormalities in Calcium ion handling and poor contractability.

Thus, the problems that result from a heart attack can spread throughout the heart and cause heart failure. In this experiment, the U of Maryland scientists injected MSCs into the sheep hearts four hours after a heart attack to determine if the stem cells could prevent the region adjacent to the dead heart cells from deteriorating.

In this experiment, bone marrow MSCs were isolated from sheep bone marrow and put through a battery of tests to ensure that they could differentiate into bone, cartilage, and fat. Once the researchers were satisfied that the MSCs were proper MSCs, they induced heart attacks in the sheep, and then injected ~200 million MSCs into the area right next to the region of the heart that died.

After 12 weeks, tissue biopsies from these sheep hearts were taken and examined. Also, the sheep hearts were measured for their heart function and structure.

The sheep that did not receive any MSC injections continued to deteriorate and showed signs of stress. The cells adjacent to the dead region expressed a cadre of genes associated with increased cell stress. Furthermore, there was increased cell death and evidence of scarring in the region adjacent to the death region. There was also evidence of Calcium ion-handling problems in the adjacent tissue and increased cell death.

On the other hand, the hearts of the sheep that had received injections of MSCs into the area adjacent to the dead region showed a reduced expression of those genes associated with increased cell stress. Also, these hearts contracted better than those that had not received stem cell injections. There was also less cell death, less scarring, and no evidence of Calcium ion-handling problems.

Changes that occur in the heart after a heart attack are collectively referred to as “remodeling.” Remodeling begins regionally, in those areas near the dead heart cells, but these deleterious changes spread to the rest of the heart, resulting in heart failure. The injections of MSCs into the area next to the dead region clearly prevented remodeling from occurring.

This pre-clinical study is a remarkable study for another reason: the MSCs used in this study were allogeneic. Allogeneic is a fancy way of saying that they did not come from the same animal that suffered the heart attack, but from some other healthy animal. Therefore, the delivery of a donor’s MSCs into the heart of a heart attack patient could potentially prevent heart remodeling.

The main problem with this experiment is that the MSCs were injected directly into the heart muscle. In humans, such a procedure requires special equipment and carries potential risks that include perforation of the heart wall, rupture of the heart wall, or further damaging the heart muscle. Therefore, if such a technology could be adapted to a more practical delivery system in humans, then certainly human clinical trials should be forthcoming.

See Yunshan Zhao, et al., “Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction.” Stem Cells Translational Medicine 2012;1:685-95.

Mesenchymal Stem Cells Found Around Blood Vessels in the Liver

Mesenchymal stem cells (MSCs) are found throughout the body and it is possible that every organ in our body has a MSC population. MSCs have the ability to differentiate into three main tissues: bone, fat and cartilage. However, the efficiency of this differentiation differs from one MSC population to another. Also, some MSCs can form smooth muscle for blood vessels and there is even evidence that MSCs can form blood vessels under certain conditions (for example, see Wingate K, Bonani W, Tan Y, Bryant SJ, Tan W. Acta Biomater. 2012 8(4):1440-9. doi: 10.1016/j.actbio.2011.12.032).

One of the places MSCs are usually found is around blood vessels. MSCs like to hang out on the outside of blood vessels in some tissues, and for this reason, MSCs are sometimes called “perivascular” stem cells.

One organ that has a stem cell population is the liver, but there is disagreement as to where they reside. Now a new publication has established that cells that hang out near blood vessels in liver are the MSC population in liver.

Eva Schmelzer from the McGowan Institute for Regenerative Medicine at the University of Pittsburgh has published a fine paper in the journal Stem Cells and Development detailing, with the help of her trusty laboratory colleagues, the characterization of liver MSCs.

Briefly, Schmelzer and her colleagues obtained fetal and adult lover tissue from tissue suppliers and minced them up, digested them with the appropriate enzymes, pushed them through cell strainers and then destroyed all the contaminating red blood cells. The remaining cells were grown in a cell culture medium. The stem cells would outgrow all the other cells, which would make their isolation and purification easy.

To purify the cells, Schmelzer’s co-workers used a technique called “flow cytometry.” When they had purified the liver MSCs, they set about characterizing them.

The liver MSCs grew quite well in culture and also grew quickly. They also expressed lots of surface proteins normally found on MSCs, confirming that they are MSCs. When gene expression experiments examined what genes these MSCs expressed, they expressed some smooth muscle genes and a several other genes enriched in cells near blood vessels. When Schmelzer examined cross sections of liver to determine where these cells are located, she found them curled up next to blood vessels.

In culture, the liver MSCs did not make very good cartilage or fat. However, they did make very good smooth muscle and bone. The efficiency of MSC differentiation tends to depend on where they were isolated. The rule of thumb is that MSCs most easily differentiate into those tissues that are closest to their own tissue of origin. Therefore, we would expect bone marrow MSCs to make better bone and cartilage than fat-based MSCs, and we would expect fat-based MSCs to make better fat than bone or liver-based MSCs. The ability of liver MSCs to be so good and making bone might be a little surprising, but when we consider that bone marrow stem cells begin their lives in the liver before they migrate to the bone marrow, perhaps this finding makes more sense.

In short, the adult and fetal liver contain a MSC population that is found on the outside of the blood vessels and these cells have an excellent capacity to make bone and smooth muscle for blood vessels. Thus liver biopsies might provide do more than provide material for diagnostic purposes – they might secure cells for regenerative purposes.

Using Tissue-Specific Mesenchymal Stem Cells to Make Insulin-Producing Beta Cells from Embryonic Stem Cells

Embryonic stem cell lines are made from four-five-day-old human embryos. At this stage of development, the embryo is a sphere of cells with two distinct cell populations; an outside layer of flat trophoblast cells and an inner clump of round inner cell mass (ICM) cells. The embryo consists of ~100 cells four days after fertilization, and ~150 cells five days after fertilization.


Embryonic stem cell (ESC) derivation involves the removal of the trophoblast cells (which are collectively called the trophectoderm) and the isolation of the ICM cells. There are several ways to remove the trophectoderm, but the most commonly-used technique is “immunosurgery,” which uses antibodies that bind to proteins on the surfaces of the trophectoderm, and serum to initiate destruction of the trophoblast cells. The isolated ICM cells are then cultured, and if they grow, they may produce an embryonic stem cell line.

Immunosurgery was first perfected by Davor Solter and Barbara B. Knowles on mouse embryos. They used an antiserum that was raised in rabbits when the rabbits were immunized against mouse spleen tissue. When mouse embryos were incubated with this antiserum plus serum from mice, all the cells of the mouse embryo died. However, if they used the rabbit antiserum and serum from guinea pig, then only the trophoblast cells were destroyed. For human embryonic stem cell derivation, the rabbits are immunized again human red blood cells, and this rabbit antiserum is used with guinea pig serum. The serum contains proteins called “complement,” which bind to cells that have antibodies attached to them and bore holes in those cells, thus destroying them.

When ICM cells are cultured, they are placed on a layer of mouse cells that have been treated with a chemical called mitomycin C to prevent them from dividing. These non-dividing cells act as “feeder cells” that keep the ICM cells from differentiating. Because ICM cells are grown on animal cells, they cannot be used for clinical purposes, since they will possess animal proteins can carbohydrates on their surfaces, which would be attacked by the patient’s immune system. However, several ESC lines have been derived without animal products, and it is possible to make ESC lines that would be safe or human use.

ESC derivation

ESC derivation results in the destruction of human embryos. There is not two ways about it. Even though there are potential ways around this problem, the majority of ESC lines were made literally over the dead bodies of very young human beings. All the rationalization in the world (the embryo is too young, too small, too inchoate, too unwanted, going to die anyway, in the wrong place at the wrong time) do not undo the fact that the embryo is a very young human being, and making an ESC line from it ends his/her life.

Getting the ESC line to differentiate in what you want it to be is another problem. If any undifferentiated cells remain after differentiation, they can cause tumors. Therefore, there is a need to ensure that differentiation is efficient and complete. To this end, Doug Melton’s lab at Harvard University has published a remarkable paper in the journal Nature that uses mesenchymal stem cells from particular organs to direct the differentiation of ESC lines.

Melton’s lab, in particular Julie M. Sneddon and Malgorzata Borowiak (say that fast five times), established 16 lines of tissue-specific mesenchymal stem cells (MSCs) from embryonic, neonatal and adult mouse intestine, liver, spleen, and pancreas and human pancreas too. Then they cultured mouse ESCs on these MSC lines to determine if they could drive the ESCs to differentiate into pancreas cells. In the embryo, pancreatic precursors express several genes in a nested, hierarchical fashion. First, they express Sox17, which is a common endodermal marker, and then pancreatic progenitors all express Pdx1. Of these pancreatic progenitors, some express Ngn3 and these will become endocrine rather than exocrine cells, and othe the Ngn3-expressing cells, a few will become beta cells that make insulin.

Melton and his co-workers tried to determine if any of these genes was up-regulated in their ESC lines if that were co-cultured with their established MSC lines. They discovered that four lines – MSC1, 2, 3, & 4, all affected gene expression when co-cultured with ESCs. MSC 1 and 2 induced and increase in Sox17 expression and MSC 3 and 4 increased the expression of Ngn3 in ESCs.

These changes in gene expression were due to increased cell proliferation of cells actually expressing these genes and not due to differential survival. Also, no combination of growth factors could achieve the same results as the accompanying MSC lines. Thus there is more going on here than the MSCs just secreting the right growth factors. The MSCs must be making contact with the ESCs and inducing them to differentiate into a particular cell type.

Next Melton and his colleagues determined if this interaction with MSCs caused the ESCs to lose their ability to self-renew. The answer was a clear “no.” Even though these ESC lines were expressing genes characteristic of endodermal or pancreatic tissue, they did not lose their ability to differentiate into pancreatic tissue when appropriately induced to do so, and they also id not lose their ability to self-renew and grow competently in culture.

In a more stringent test, these ESCs that had been grown on tissue-specific MSCs were implanted into mice. As Melton points out in the paper, the “most efficient published protocols for in vitro differentiation of pluripotent cells to beta-cells yield only a small percentage (typically 0-15%) of insulin-positive cells, and these do not secrete insulin in a glucose-responsive manner.” Could the MSC-conditioned ESCs do any better?

Before implantation, the ESCs were differentiated into endodermal progenitors (Sox17-expressing cells), and co-cultured with MSCs for at least 3-7 passages. Then they were differentiated into beta cells and transplanted into mice. There were a few important controls that were used; Just saline, implantations of MSCs alone, and ESCs that had been differentiated into beta cells, but had never been passaged on MSCs. Finally, human pancreatic islets were used as a positive control.

The results were interesting to say the least. The saline and MSC alone implantations showed no insulin production with or without glucose. Likewise the human pancreatic islets made insulin in a glucose-dependent manner (no surprise there). The ESC-derived beta cells that had never been passaged on MSCs made insulin, and even showed some ability to respond to glucose and make more insulin after glucose ingestion. However, the beta cells derived from ESCs that had been passaged on MSCs made insulin in a glucose-dependent manner. The experiment produced a wide range of variability since the number of transplanted cells differed between each trial, but the implanted beta cells derived from ESCs passaged on tissue-specific MSCs definitely performed the best, and even did as well or better than the implanted human beta cells in some cases.

a, Schematic depicting implantation of human ESC-derived progenitors. b, Immunofluorescence staining of human ESC-derived endoderm, passaged seven times on mesenchyme and engrafted for 3 months (top panel) or further differentiated to Pdx1+ stage and then engrafted for 2 months (bottom panel). c, Glucose-tolerance test of animals implanted with PBS or mesenchyme only, human islets or Pdx1+ pancreatic progenitors derived from unpassaged (P0), or passaged (P4 or P7) human endoderm. d, Fasting- and glucose-induced (45 min glucose) plasma human C-peptide levels. Pairs of bars represent two time points per animal; data represent mean of two technical replicates ± s.d.
a, Schematic depicting implantation of human ESC-derived progenitors. b, Immunofluorescence staining of human ESC-derived endoderm, passaged seven times on mesenchyme and engrafted for 3 months (top panel) or further differentiated to Pdx1+ stage and then engrafted for 2 months (bottom panel). c, Glucose-tolerance test of animals implanted with PBS or mesenchyme only, human islets or Pdx1+ pancreatic progenitors derived from unpassaged (P0), or passaged (P4 or P7) human endoderm. d, Fasting- and glucose-induced (45 min glucose) plasma human C-peptide levels. Pairs of bars represent two time points per animal; data represent mean of two technical replicates ± s.d.

Melton notes at the end of his paper that this technique worked rather well for coaxing ESCs to form pancreatic derivatives, but it could very well be applicable to other systems as well. Also, it could probably work with induced pluripotent stem cells, which have many (though not all) of the characteristics of ESCs and can be made without killing human embryos. Thus another technique for increasing ESC differentiation seems to be on the table.

A Co-culture System Makes Better Cartilage for Tissue Replacement

At joints, the bones are covered with cartilage to act as a shock absorber. Articular cartilage, or cartilage at joints, is usually characterized by very low friction, high wear resistance, but very abilities to regenerate. Articular cartilage is responsible for much of the compressive resistance and load bearing qualities of joints, and without it, even activities as simple and walking is too painful. Osteoarthritis is a condition that results from cartilage failure, and limits the range of joint motion, increases the bone damage and also causes a respectable amount of pain. When the cartilage of the articular surface erodes, the bone is exposed and grinding of the bone creates bone spurs, extensive inflammation and pain.

Treating osteoarthritis requires that one make new cartilage that has similar properties as articular cartilage. Unfortunately, mesenchymal stem cells that are differentiated into cartilage making cells (chondrocytes) and implanted into the knee tend to make fibrocartilage, which is different than the hyaline cartilage that composes articular cartilage. Fibrocartilage does not possess the high-wear resistance characteristics of hyaline cartilage and it tends to erode rather rapidly after formation. Therefore, directing mesenchymal stem cells (MSCs) to form proper cartilage is a genuine challenge.

A paper that appear in Stem Cell Translational Medicine from Gilda A. Barabino, who is a faculty member at the Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology and Emory University, examines a technique to coax MSCs to make articular cartilage.

As Barbino points out, traditional protocols that direct MSCs to differentiate into chondrocytes uses culture systems of MSCs that have been treated with various growth factors, such as transforming growth factor-β. Unfortunately, these culture systems tend to fall short in meeting the needs of clinical applications, largely because they yield terminally differentiated cells that enlarge and then form bone.

In this study Barbino and her co-workers co-cultured bone marrow-derived MSCs with juvenile articular chondrocytes. The rationale is that the MSCs would receive just the right growth factors in just the right concentrations and at the right time to drive MSC cartilage formation. Physical contact between cells can also do a better job of driving them to differentiate into various cells types rather than simply treating them with growth factors.

Barbino and others discovered that an initial chondrocyte/MSC ratio of 63:1 worked the best and the MSCs form chondrocytes that had the right cells shape, behavior, and characteristics of articular chondrocytes.

Next, Barbino and her team grew the MSCs in a three-dimensional agarose system. Three-dimensional systems are generally thought to more realistically recapitulate the cartilage-making system present at joints. In this 3-D culture system, when co-cultured with juvenile articular chondrocytes, bone marrow MSCs develop into robust neocartilage that was structurally and mechanically stronger than the same cultures that only contained chondrocytes.

There was another advantage to this culture system; cultured MSCs that are induced to form cartilage tend to cease all expression of a surface protein called CD44, which is an important regulator in cartilage biology. However, when cultured in the 3-D culture, the MSCs retained the expression of CD44, which suggests that these co-cultured MSCs, which cultured in a 3-D culture system form chondrocytes that make superior articular cartilage, but retain CD44, which allows cartilage maintenance.

This shows that making articular cartilage from MSCs is probably possible and only requires the right culture system. Also, co-culturing MSCs with articular chondrocytes in a 3-D culture system might be one of the better culture systems for developing clinically relevant cartilage for tissue replacements.

Making Artificial Tissues With Bioprinters

Brian Derby from the University of Manchester is using inkjet technology to distribute cells onto scaffolds that are shaped as a particular organ. Inkjet and laserjet technologies can build three-dimensional scaffolds that are coated with cells that will grow into the scaffold, assume its shape and degrade the scaffold, leaving only the tissue in its place.

This type of technology, which involves the simultaneous placement of biodegradable scaffold and cells in a three-dimensional structure that resembles that of an organ is called additive manufacture and it might very well be the future of replacement organ production.  Additive manufacture recreates the biological structure in a three-dimensional, digital image, from which two-dimensional, digital slices are taken and fashioned one layer at a time.  The summation of all the digital slices eventually produces a three-dimensional structure.

Inkjet technology dispenses the material that makes the scaffold in very small droplets that quickly solidify.  The materials is loaded into an actual inkjet printer cartridge that is sprayed onto the surface.  More droplets are placed on top of previous droplets in a very specific pattern and this repetitive distribution of droplets develop into a pattern that is very complex and forms a scaffold that nicely mimics the conditions inside the body.  The scaffold also provides a surface the for cells to adhere, grow and thrive.  The scaffold and its internal structure control the behavior and maintain the health of the cells embedded in the scaffold.  This method of distributing cells onto a surface through a printer is called “bioprinting.”

In his article, published in the journal Science, Derby examines experiments in which porous structures are made by means of bioprinting.  Bioprinting uses inkjet and laserjet technologies to distribute cells or molecules onto a surface in a desired pattern.  In the case of porous structures, cells interweave throughout the scaffold and such cell-encrusted scaffolds can be placed in the body to encourage cell growth.  Depending on the composition of the scaffold and the cells embedded in it, the scaffold can become a part of the body or the cells will dissolve it.   Such a treatment can help heal patients with particular injuries such as cavity wounds.

Bioprinted cells can also be deposited onto scaffolds with various other chemicals, such as hormones, growth factors, or small molecules that influence the behavior of the cells.  The inclusion of such molecules with the scaffold can coax cells to differentiate into distinct cell types, such as, for example, bone- or cartilage-producing cells.

Cells do suffer some damage during bioprinting, and the rule of thumb is the more energy is used to deposit the cells onto the scaffold, the lower the viability of the cells after bioprinting.  To deposit and pattern cells in a scaffold there are three techniques that are used:  inkjet printing, microextrusion, which is also known as filament plotting, and laser forward transfer.  Bioprinting has probably the highest viability rates, and that has come after the techniques have been precisely worked out to ensure a minimum of damage.  Microextrusion shows extremely variable rates of cell survival after the cells are deposited.  Laser forward transfer suffers from the need for higher energy lasers to more precisely and efficiently deposit the cells, but this same higher energy kills off the cells.

Even though this technology has come a long way, it has a way to go before it is ready for the clinic.  Scaffolds are being used in clinical trials, but scaffold synthesis suffers from inconsistency, and until a consistent high-quality is delivered, scaffold production will not be ready for commercial production.

Despite these caveats, there have been some successes.  For example, D’Lima and others used an solution of chemicals in water (poly(ethylene glycol) dimethacrylate to be exact) that also contained cartilage-making cells (chondrocytes).  They printed this suspension a bone defect in a cultured bone and then used a chemical not unlike what dentists use to harden tooth plastic called a photoinitiator.  Such chemicals crosslink and bond together in response to particular wavelengths of light, and D’Lima used light to crosslink the chemicals to make a wet gel that contained the cells.  After several days, this printed structure appeared to have integrated into the surrounding tissue.  This experiment demonstrates that this technology is at least feasible.  The hanging issue is the toxicity of the photoinitiator chemicals to cells (X. Cui, et al Tissue Eng. A 18, 1304 (2012).  However, this has been studied, and it turns out the susceptibility to these chemicals is very cell type-specific.  Thus, picking the right photoinitiator could potentially make this technique rather safe (see C. G. Williams, et al Biomaterials 26,1211 2005).

(A) Schematic of bioprinting a cartilage analog structure, combining inkjet printing with a poly(ethylene glycol) dimethacrylate (PEGDMA) solution containing cells in suspension with a simultaneous photopolymerization process. (B) Light microscopy image of cell-containing polyethylene hydrogel printed into a defect formed in an osteochondral plug (scale bar, 2 mm). After culture, the cells within the printed material express ECM similar to those in the adjacent tissue

Scaffolds, however, can also be used to make external tissues, for example, skin patches.  Derby is working with ear, nose, and throat surgeons at the Manchester Royal Infirmary.  His goal is to use bioprinting to make patches that can be implanted into the inside of the nose or throat.

Derby explains: “It is very difficult to transplant even a small patch of tissue to repair the inside of the nose or mouth.  Current practice, to transplant the patient’s skin to these areas, is regarded as unsatisfactory because they transplants do not possess mucous generating cells or salivary glands.  We are working on techniques to print sheets of cells that are suitable for implantation in the mouth and nose.”

Derby hopes that someday bioprinting can be used to grow tumors in realistic cultures that will make superior models for drug testing and drug development.

TIMP3 Secreted by Mesenchymal Stem Cells Protects the Blood Brain Barrier After a Traumatic Brain Injury

Mesenchymal stem cells (MSCs) are found in multiple tissues and locations throughout our bodies, and they have the ability to differentiate into bone, fat, cartilage, and smooth muscle. MSCs also have the ability to suppress unwanted immune responses and inflammation. Therefore, MSCs are prime candidates for regenerative medical treatments.

MSCs have been used to experimentally treat traumatic brain injury (for example, Galindo LT et al., Neurol Res Int 2011;2011:564089). One of the main concerns after traumatic brain injury is damage to the blood brain barrier (BBB). BBB damage allows inflammatory cells to access the brain and further damage it. Therefore, healing the damage to the BBB or protecting the BBB after a traumatic brain injury is vital to the brain after a traumatic brain injury.

After a traumatic brain injury, the vascular system suffers damage and begins to leak. When blood leaks into tissues, it tends to irritate the tissues and damage them. MSCs release a soluble factor known as TIMP3 (tissue metalloproteinase-3) that degrades blood-based proteins known to cause damage to tissues when blood vessels leak. TIMP3 production by MSCs can also protect the BBB from degradation after a traumatic brain injury.

Researchers from the University of Texas Health Sciences Center, UC San Francisco, and two biotechnology companies have examined the protective role of MSCs and one particular protein secreted by MSCs in protecting the BBB after traumatic brain injury.

Shibani Pati, from UC San Francisco, and his collaborators from the University of Texas, Houston, MD Anderson Cancer Center, Amgen, and Blood Systems Research Institute (San Francisco) used MSCs to staunch the increased permeability the BBB after a traumatic brain injury.

They used a mouse model in these experiments and induced traumatic brain injuries in these mice. Then they gave MSCs to some, and soluble TIMP3 to others, and buffer to another group as a control. They discovered that the MSCs mitigated BBB damage after a traumatic brain injury. However, they also found that soluble TIMP3 could also protect the BBB approximately as well as MSCs. This suggested that the TIMP3 secretion by MSCs is the main mechanism by which MSCs protect the BBB after a traumatic brain injury.

To test this hypothesis, Pati and his colleagues administered MSCs to mice that had experienced traumatic brain injury, but they also co-administered a soluble inhibitor to TIMP3. They discovered that this inhibitor completely abolished the ability of MSCs to protect the BBB after a traumatic brain injury. They also found that the main target of TIMP3 was vascular endothelial growth factor. Apparently after a traumatic brain injury, massive release of vascular endothelial growth factor causes the breakdown of BBB structures. TIMP3 degrades vascular endothelial growth factor, which prevents BBB breakdown.

These findings suggest that administration of recombinant proteins such as TIMP3 after a traumatic brain injury can protect the BBB and decrease brain damage. Clinical trial anyone?

Highly Efficient Method for Converting Blood Stem Cells into Induced Pluripotent Stem Cells Without Viruses

A research group from Johns Hopkins University has designed a protocol that reliably converts stem cells from umbilical cord blood into a primitive stem cell state. From this primitive state, these cells can differentiate into any other type of cell in the body.

This paper was published in the August 8th issue of Public Library of Science (PLoS), and serves as the second publication in an ongoing effort to efficiently and consistently convert umbilical cord blood stem cells and other types of stem cells into stem cells that are usable for use in clinical and research settings in place of human embryonic stem cells, according to Elias Zambidis, M.D., Ph.D., who is an assistant professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center.

Zambidis said: “Taking a cell from an adult and converting it all the way back to the way it was when that person was a 6-day-old embryo creates a completely new biology toward our understanding of how cells age and what happens when things go wrong, as in cancer development.”

The first paper that is sometimes designated ‘Chapter One‘ of this work was published last spring in PLoS One. In this paper, Zambidis’ group described the successful use of a method that safely transformed several different types of human pluripotent stem cells into heart muscle cells. In the latest experiments, Zambidis and his colleagues describe methods that convert umbilical cord blood stem cells into induced-pluripotent stem cells (iPS), which are adult or fetal cells reprogrammed to an embryonic like state.

According to Zambidis, he and his team developed a “super-efficient, virus-free” method for making iPS cells. This overcomes some troubling difficulties for those scientists who work with iPS cells; namely, the vast inefficiency of making iPS cells from adult cells and the use of mutation-causing viruses to introduce those genes into adult cells required to convert adult cells into iPS cells. Generally, out of hundreds of blood cells, only one or two typically revert into iPS cells. However, with Zambidis’ method, 50-60% of blood cells were engineered into iPS cells.

To circumvent the use of viruses to deliver genes, Zambidis’ team used plasmids, or small circles of DNA that replicate briefly inside cells and then degrade. By using plasmids, the cells receive the genes required to drive adult cells into the iPS state, but because these genes are only required transiently, the plasmids do their job and then go away. Therefore, the production of mutations by viral DNAs that insert themselves into the host cell genome is not a problem with this method from Zambidis’ laboratory.

In order to introduce the genes into the cells, Zambidis’ team used a technique called electroporation. They treated the umbilical cord blood cells with the plasmids and then delivered an electrical pulse to the cells, which made tiny holes in the surface through which the plasmids could slip to the cell interior. Once inside, the plasmids triggered the cells to revert to a more primitive cell state. After genetic engineering, the blood cells were also given an additional new step in which they were stimulated with their natural bone-marrow environment. To do this, the Johns Hopkins team took some of the treated cells in a dish alone, and cultured them together with irradiated bone-marrow cells.

When iPS cells made from umbilical cord blood were compared to iPS cells made from hair cells and from skin cells, they found that the most superior iPS cells came from those made from blood stem cells treated with just four genes and cultured with the bone marrow cells. These cells reverted to a primitive stem cell state within seven to 14 days. Their techniques also successfully converted blood stem cells from adult bone marrow and from circulating blood into iPS cells.

In ongoing studies, Zambidis and colleagues are testing the quality of their newly formed iPS cells. They are also interested in the ability of these iPS cells to differentiate into other cell types, as compared with iPS cells made by other methods. These efficient methods to produce virus-free iPS cells will hopefully speed research to develop stem cell therapies that use nearly all cell types, and may provide a more accurate picture of cell development and biology.

Prosthetic Retina Restores Sight In Mice

A pair of neuroscientists have designed a prosthesis that partially restores sight in mice. To get this device to work, they had to decipher the code by which the retina tells the brain what the eye has seen. They hope to adapt the device to test in human patients.

Globally, some 20 million people blind from retinal disease that cause degeneration of retinal cells. The retina is a thin tissue at the back of the eye that actually is composed of multiple cell layers that help convert electromagnetic energy in the form of visible light into a neural signal. Presently, there is only one prosthetic device that has been approved for treatment of such conditions. This device consists of an array of surgically implanted electrodes that directly stimulate the optic nerve in response to light. It allows patients to discern edges and letters, but patients cannot recognize faces or perform many everyday tasks.

Sheila Nirenberg, a physiologist at the Weill Medical College at Cornell University in New York, thinks that the reason the present prosthetic devices work poorly is that they do not properly code the light energy into a form that the brain can interpret. The retina contains several layers of nerves that seem to translate light energy into encoded neural signals. According to Nirenberg, “The thing is, nobody knew the code.” Without knowing this retinal coding system, Nirenberg believes that visual prostheses will never be able to create images that the brain can easily recognize.

In light of this, Nirenberg and her student, Chethan Pandarinath, have devised a code and developed a device that uses it to restore some sight in blind mice.

They began their work by injecting nerve cells into the retinas of mice with a genetically engineered virus. This virus had been designed to insert a gene that causes the cells to produce a light-sensitive protein normally found in algae. When a beam of light was then projected into the eye, the algal protein triggered the nerve cells to send a signal to the brain. By engineering the neural cells in this fashion, the ganglion cells that normally receive the message from the photoreceptors cells, which directly respond to light, sent neural signals directly to the brain and performs in a manner similar to healthy rod and cone cells.

This experiment is not unique, since Zhuo-Hua Pan’s laboratory at Wayne State University School of Medicine had published a similar result in 2006 (here). However, Nirenberg and Pandarinath went a step further. Instead of feeding the visual signals directly into the eye, they processed these signals using a code that they had developed by watching how a healthy retina responds to stimuli. After the ganglion cells that received the encoded input, the mice were able to track moving stripes, which is something that they hadn’t been able to do before. Then Nirenberg and Pandarinath examined the neural signals that the mice were producing and they a used a different, ‘untranslate’, code to determine what the brain would have been seeing. The encoded image was clearer and more recognizable than the non-encoded one (see image).

A prosthetic retina that can translate an image into neural signals was tested using a picture of a baby’s face. A is the original image. B is the image after it passes through the coding software. C is after it has been processed by the retinal cells. D is the processed image without coding.

Researchers attempting to design visual prostheses have debated the importance of encoding. Some think that it will be crucial, but others think the brain can adapt to an unprocessed signal. James Weiland, an ophthalmologist at the University of Southern California in Los Angeles, notes that Nirenberg and Pandarinath have shown that encoding provides an advantage, but how effective it is in human patients is completely unknown until the technique is tried out in people. Weiland probably speaks for the entire field when he commented that “You can’t say for sure until you have the patient telling you ‘yes I see it. It’s better when you do that.'”

Nirenberg hopes to test her system in human trials soon. The encoding is relatively simple and can be done by a microchip. The combination of the microchip with a small video camera could fit onto a pair of glasses. The camera would record a signal and the encoder would then flash it directly onto the genetically treated nerve cells in the eye. If this prosthesis works, the technique is simple enough that it could be done in a doctor’s office. “We would like to [try it] in patients in the next one or two years,” she says.

Getting Genes into Stem Cells Without Viruses

Genetic engineering of cells and, in particular, of stem cells has the ability to adjust the functional capacities of cells. Unfortunately, genetically engineering cells requires the use of viruses that introduce genes into cells and, by doing so, produce mutations in cells.

However, there are new ways to put genes into cells without the use of viruses. By surrounding DNA that encodes the genes you want to put into cells with positively-charged lipids, you have made a structure called a liposome. Liposomes can fuse with the membranes of cells and deliver the genes to cells without viruses that can cause mutations.

A paper that has appeared in the journal Stem Cells and Development examined the use of liposomes to introduce genes into blood cell-making stem cells (HSCs). They used commercially-available systems to transfer genes into these stem cells, but they found that their own lab-designed system did a better job than the commercially-available systems.

The lead author of this paper is Hilal Gul-Uludag and the senior author is Jie Chen from the University of Alberta in Edmonton, Alberta, Canada. In this paper, Chen’s research group isolated blood cell-making stem cells from umbilical cord blood. Then they used liposomes to insert the CXCR4 gene. The CXCR4 gene encodes a receptor for “stromal cell-derived factor-1alpha” (SDF-1alpha). When cells bind to SDF-1alpha, they move towards the source of SDF-1alpha.

Interestingly, one of the best sources of SDF-1alpha is the bone marrow. If HSCs could be engineered to make CXCR4, then they would readily move into the bone marrow. This means that implanted HSCs would only need to be introduced into the peripheral blood and not into the bone. This would increase the efficiency of bone marrow or umbilical cord transplants.

Chen’s group showed the feasibility of such experiments, and that these treatments are not toxic in any way to the HSCs. Thus, such a strategy could potentially increase the efficiency of bone marrow and umbilical cord blood transplantation.

How Stem Cells Make New Skin Cells Throughout Life

Beneath the upper epidermal layers of our skin lies a layer of stem cells and their progeny (human epidermal progenitor cells) that continually make new skin throughout our lifetime. How these stem cells manage to form skin and not some other structure is still poorly understood, but a new study from the University of San Diego School of Medicine in the laboratory of George L. Sen has pulled back the curtain on this vital process.

Sen and his colleagues have examined a component of the machinery of the cell known as the “exosome.” The term exosome is confusing because it refers to two different entities. Exosomes are vesicles secreted by cells that are loaded with proteins and RNA molecules that the cell wants to dump (Kooijmans, et al., Int J Nanomedicine. 2012; 7: 1525–41). Exosomes are used by cells to export materials to other others cells. Cells also use exosomes to regulate processes, since by ridding themselves of proteins and RNAs that direct particular processes, effectively shuts those processes down. However, exosome also refers to a complex of proteins that are involved in 3′–5′ exonucleolytic degradation. This exosome consists of ~11 proteins that degrade RNAs and regulate processes.

In skin-based stem cells, the exosome (RNA degradation machinery) functions in skin stem cells and provides one of the main mechanisms by which stem cells stay stem cells and skin cells stay skin cells. Exosomes and their targets may help point the way to new drugs or therapies for not just skin diseases, but other disorders in which stem and progenitor cell populations are affected.

Stem cells can divide throughout their lifetime, and their progeny can differentiate to become any required cell type. The progeny of stem cells, progenitor cells, have more limited developmental capabilities, and are only able to divide only a fixed number of times and form a few distinct cell types. When it comes to skin, progenitor and stem cells deep in the epidermis constantly produce new skin cells called keratinocytes that gradually rise to the surface where they will mature, die, and be sloughed off.

Exosomes degrade and recycle different RNA molecules, such as messenger RNAs that wear out or that contain errors. Such errors would cause the production of junk protein, and this would be deleterious to the cell.

According to Sen: “In short, the exosome functions as a surveillance system in cells to regulate the normal turnover of RNAs as well as to destroy RNAs with errors in them.” Sen and his colleagues discovered that in the epidermis the exosome functions to target and destroy mRNAs that encode for transcription factors that induce differentiation. One of the targets of the exosome in epidermal progenitor cells is a transcription factor called GRHL3. GRHL3 promotes the expression of genes necessary for skin cell differentiation. Routine destruction of GRHL3 keeps epidermal progenitor cells undifferentiated. When the epidermal progenitor cells receive signals to differentiate, the progenitor cells down-regulate the expression of certain subunits of the exosome, and this leads to higher levels of GRHL3 protein. The increase in GRHL3 levels promotes the differentiation of the progenitor cells to skin cells.

“Without a functioning exosome in progenitor cells,” said Sen, “the progenitor cells prematurely differentiate due to increased levels of GRHL3 resulting in loss of epidermal tissue over time.” Sen also noted that these findings could have particular relevance if future research determines that mutations in exosome genes are linked to skin disorders or other diseases.

“Recently there was a study showing that recessive mutations in a subunit of the exosome complex can lead to pontocerebellar hypoplasia, a rare neurological disorder characterized by impaired development or atrophy of parts of the brain,” said Sen. “This may potentially be due to loss of progenitor cells. Once mutations in exosome complex genes are identified in either skin diseases or other diseases like pontocerebellar hypoplasia, it may be possible to design drugs targeting these defects.”

Plant Virus Speeds Bone Growth from Stem Cells

Sometimes great scientific discoveries are the result of serendipity, The German organic chemist August Kekulé supposedly came upon the structure of the chemical benzene because of a day-dream in which he saw a snake biting its own tail. This story illustrates that even brilliant scientists conceive of some of their greatest ideas in ways that are accidental.

Today’s discovery is a stem cell version of August Kekulé’s day-dream. Researchers at the University of South Carolina have been interested in growing bone from stem cells. In order to get the cells to form bone, Qian Wang, Robert L. Sumwalt Professor of Chemistry at the University of South Carolina, grew mesenchymal stem cells (MSCs) from bone marrow on plastic culture dishes. However, he wanted to test the ability of different surfaces to influence bone formation by MSCs. In order to compare each surface he tested with something that seemed rather innocuous, Wang decided to use plant viruses.

Plant viruses such as turnip yellow mosaic virus are completely harmless to animal cells and they can be isolated in gram quantities from cabbages, which makes them very inexpensive to work with. Wang decided to coat his culture dishes with these plant viruses in order to have something that was a sort of ground-zero surface that did not do anything to the cells. Except there was one problem: The plant virus-coated surfaces helped the stem cells make bone faster than anything else Wang’s group examined.

This gave Wang an idea. When bones break in our bodies, stem cells make new bone at the site of the break in order to help the bones reform and heal themselves. The process can take some time and people with broken bones are usually incapacitated for some time. Wang wondered, “what if we could make that healing process go faster?”

Wang explains: “With a broken femur, a leg, you can be really incapacitated for a long time. In cases like that, they sometimes inject a protein-based drug, BMP-2 [bone morphogen protein-2], which is very effective is speeding up the healing process. Unfortunately,, it’s very expensive and can also have some side effects.” One of those side effects is an increased risk of tumors.

By coating glass slides with turnip yellow mosaic viruses (TYMVs) or tobacco mosaic viruses (TMVs), Wang and his colleagues found that the stem cells did not take nearly as long to form bone in culture.

Since making that discovery four years ago, Wang has been trying to determine what is it about the viruses that cause the MSCs to make bone so effectively. It turns out that the viruses form a specific topography on the glass slides and this topography forms a kind of “easy chair” for the MSCs. The next question was, “Could this easy chair be made into more of a Sleep Comfort Bed for the MSCs?” Could they improve it?

Wang and his group set about chemically modifying the surfaces of the viruses and binding things to them in order to stabilize the interaction of the viruses with the MSCs and the slide.

The results were astounding. With the right concoction of plant viruses coating the glass slides, the right molecules bound to the plant viruses, and the right culture medium recipe, Wang’s group found that they could induce MSCs to form bone in two days. The cells are also make many proteins that are specific to bone formation.

According to Wang: “What we’ve seen could prove very useful, particularly when it comes to external implants in bones. With those, you have to add a foreign material, and knowing that a coating might increase the bone growth process is clearly beneficial. But more importantly, we fell we’re making progress in a more general sense in bone engineering. We’re really showing the direct correlation between nanotopography and cellular response. If our results can be further developed, in the future you could use titanium to replace the bone and you might be able to use different kinds of nanoscale patterning on the titanium surface to create all kinds of different cellular responses.”

In many ways this work is just the beginning of what will almost certainly become a remarkable advance in bone engineering.

Platelet-Lysate Bioactive Scafold for Tissue Engineered Cartilage

Cartilage replacement at joints represents a tremendous challenge for regenerative medicine. While growing cartilage in culture is possible, scaling this technology up to generate enough high-quality articular cartilage (the kind of cartilage found at joints), is still a distinct challenge. To date, stem cell treatments can heal small breaches in cartilage, but reconstructing large lesions is still not possible. In general, cartilage at joints has very poor healing properties, and therefore, is a major challenge in orthopedics.

A major improvement in therapeutics is the use of a technique called “autologous chondrocyte implantation” or ACI. ACI involves the delivery of healthy cartilage-making cells (chondrocytes) from the patient’s own body after these cells have been grown and expanded in culture. In order to coax these cartilage-making cells to make cartilage, special scaffolds are used that provide a three-dimensional matrix upon which the chrondrocytes grow and form cartilage. These 3-D scaffolds are essential to keep the chondrocytes differentiated and making cartilage.

One of the most promising types of scaffolds for making cartilage are “bioactive 3D scaffolds.” These types of scaffolds can deliver growth factors and other molecules to the chrondrocytes and boost their growth and cartilage production.

In a recent publication, Andrei Moroz and colleagues in the Extracellular Matrix Laboratory at the Botucatu Institute of Biosciences, São Paulo State University, Brazil, have used mesenchymal stem cells (MSCs) from rabbit bone marrow and differentiated them into chondrocytes. This allowed them to use stem cells from bone marrow instead of harvesting cartilage from the joints, which can be very painful and deleterious to the joint. The main innovation in this paper was the use of a platelet-lysate-based 3D bioactive scaffold to support the chondrogenic differentiation and maintenance of MSCs.

MSCs from adult rabbit bone marrow were isolated, characterized, and grown in 60 microliters of platelet lysate from rabbit blood. Platelets are very small cells from circulating blood that assist in the formation of clots that staunch bleeding after a blood vessel in damaged. Platelets are easy to isolate from circulating blood and the rabbit platelet-lysate clot scaffold was maintained is a standard tissue culture medium (Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12) that was supplemented with other molecules known to induce cartilage formation in MSCs. After three weeks in culture, the MSCs were examined in detail. Not only were they nice and round, but they were filled with cartilage-specific molecules, and clumped together like chondrocytes.

According to this research group, they are on to something with this platelet-lysate bioactive scaffold. It provided a suitable system for culturing MSCs and allowed them to make lots of cartilage. The scaffold also was easy to make, and maintained the MSCs in a cartilage-making state without causing cell death or stressing the cells. Therefore, it might provide an alternative to autologous chondrocyte implantation. The next steps in this research will be to use this engineered cartilage to repair damaged joints to see if the cartilage made by cells embedded in platelet-lysate 3D bioactive scaffolds can act as functional cartilage.

For the article see Andrei Moroz, et al., Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering.  Platelets. 2012.