Hematopoietic Stem Cells Use a Simple Heirarchy

New papers in Science magazine and the journal Cell have addressed a long-standing question of how the descendants of hematopoietic stem cells in bone marrow make the various types of blood cells that course through our blood vessels and occupy our lymph nodes and lymphatic vessels.

Hematopoietic stem cells (HSCs) are partly dormant cells that self-renew and produce so-called “multipotent progenitors” or MPPs that have reduced ability to self-renew, but can differentiate into different blood cell lineages.

The classical model of how they do this goes like this: the MPPs lose their multipotency in a step-wise fashion, producing first, common myeloid progenitors (CMPs) that can form all the red and white blood cells except lymphocytes, or common lymphoid progenitors (CLPs) that can form lymphocytes (see the figure below as a reference). Once these MPPs form CMPs, for example, the CMP then forms either an MEP that can form either platelets or red blood cells, or a GMP. which can form either granulocytes or macrophages. The possibilities of the types of cells the CMP can form in whittled down in a step-by-step manner, until there is only one choice left. With each differentiation step, the cell loses its capacity to divide, until it becomes terminally differentiated and becomes platelet-forming megakarocyte, red blood cell, neutrophil, macrophage, dendritic cells, and so on.


These papers challenge this model by arguing that the CMP does not exist. Let me say that again – the CMP, a cell that has been identified several times in mouse and human bone marrow isolates, does not exist. When CMPs were identified from mouse and human none marrow extracts, they were isolated by means of flow cytometry, which is a very powerful technique, but relies on the assumption that the cell type you want to isolate is represented by the cell surface protein you have chosen to use for its isolation. Once the presumptive CMP was isolated, it could recapitulate the myeloid lineage when implanted into the bone marrow of laboratory animals and it could also produce all the myeloid cells in cell culture. Sounds convincing doesn’t it?

In a paper in Science magazine, Faiyaz Notta and colleagues from the University of Toronto beg to differ. By using a battery of antibodies to particular cell surface molecules, Notta and others identified 11 different cell types from umbilical cord blood, bone marrow, and human fetal liver that isolates that would have traditionally been called the CMP. It turns out that the original CMP isolate was a highly heterogeneous mixture of different cell types that were all descended from the HSC, but had different developmental potencies.

Notta and others used single-cell culture assays to determine what kinds of cells these different cell types would make. Almost 3000 single-cell cultures later, it was clear that the majority of the cultured cells were unipotent (could differentiate into only one cell type) rather than multipotent. In fact, the cell that makes platelets, the megakarocyte, seems to derive directly from the MPP, which jives with the identification of megakarocyte progenitors within the HSC compartment of bone marrow that make platelets “speedy quick” in response to stress (see R. Yamamoto et al., Cell 154, 1112 (2013); S. Haas, Cell Stem Cell 17, 422 (2015)).

Another paper in the journal Cell by Paul and others from the Weizmann Institute of Science, Rehovot, Israel examined over 2700 mouse CMPs and subjected these cells to gene expression analyses (so-called single-cell transriptome analysis). If the CMP is truly multipotent, then you would expect it to express genes associated with lots of different lineages, but that is not what Paul and others found. Instead, their examination of 3461 genes revealed 19 different progenitor subpopulations, and each of these was primed toward one of the seven myeloid cell fates. Once again, the presumptive CMPs looked very unipotent at the level of gene expression.

One particular subpopulation of cells had all the trappings of becoming a red blood cell and there was no indication that these cells expressed any of the megakarocyte-specific genes you would expect to find if MEPS truly existed. Once again, it looks as though unipotency is the main rule once the MPP commits to a particular cell lineage.

Thus, it looks as though either the CMP is a very short-lived state or that it does not exist in mouse and human bone marrow. Paul and others did show that cells that could differentiate into more than one cell type can appear when regulation is perturbed, which suggests that under pathological conditions, this system has a degree of plasticity that allows the body to compensate for losses of particular cell lineages.

A model of the changes in human My-Er-Mk differentiation that occur across developmental time points. Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.

A model of the changes in human My-Er-Mk differentiation that occur across developmental time points.
Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.

Fetal HSCs, however, are a bird of a different feather, since they divide quickly and reside in fetal liver.  Also, these HSCs seem to produce CMPs, which is more in line with the classical model.  Does the environmental difference or fetal liver and bone marrow make the difference?  In adult bone marrow, some HSCs nestle next to blood vessels where they encounter cells that hang around blood vessels known as “pericytes.”  These pericytes sport a host of cell surface molecules that affect the proliferative status of HSCs (e.g., nestin, NG2).  What about fetal liver?  That’s not so clear – until now.

In the same issue of Science magazine, Khan and others from the Albert Einstein College of Medicine in the Bronx, New York, report that fetal liver also has pericytes that express the same cell surface molecules as the ones in bone marrow, and the removal of these cells reduces the numbers of and proliferative status of fetal liver HSCs.

Now we have a conundrum, because the same cells in bone marrow do not drive HSC proliferation, but instead drive HSC quiescence.  What gives? Khan and others showed that the fetal liver pericytes are part of an expanding and constantly remodeling blood system in the liver and this growing, dynamic environment fosters a proliferative behavior in the fetal HSCs.

When umbilical inlet is closed at birth, the liver pericytes stop expressing Nestin and NG2, which drives the HSCs from the fetal liver to the other place were such molecules are found in abundance – the bone marrow.

These models give us a better view of the inner workings of HSC differentiation.  Since HSC transplantation is one of the mainstays of leukemia and lymphoma treatment, understanding HSC biology more perfectly will certainly yield clinical pay dirt in the future.


Freezing Amnion-Derived Stem Cells in Xeno-Free Media Feasible

Amnion-derived stem cells show a dizzying set of regenerative properties, at least in the laboratory (see Miki T, Stem Cell Res Ther 2011, 2:25; Miki T, Grubbs B, J Obstet Gynaecol Res 2014, 40:360-8). Bringing these cells into the clinic will definitely take a lot more work, but a new paper examines a culture system that does not use any animal products. Such culture systems (known as “xeno-free) are vitally important if stem cells are going to be used in human trials.

When stem cells are grown in culture, typically serum from animal blood is used to provide the cells with the growth factors and things they need to kick the cells into the growth phase. However, occasionally, some animal-based products have animal viruses that can infect human cells with unpredictable consequences (see Karlsson JOM, Toner M, Biomaterials 1996, 17:243-56). Also, cells grown in animal-based culture media can have animal proteins around them that are very hard to get rid of. Implanting such cells into a human patient would cause their immune system to react against the animal proteins. Such immune responses might result in something as innocuous as itching at the site of injection or as dangerous as anaphylactic shock. Therefore, growing stem cells under xeno-free conditions is important for many stem cell applications.  Expanding and preserving stem cells under so-called Good Manufacturing Pracctices (GMP) is essential for their use in human patients.

However, now we have just opened another can of worms. If all the characterizations of stem cells have been under culture conditions that utilize animal-based culture media, will the cells have the same capabilities if grown under xeno-free conditions?

Herein is the reason why this paper by Toshio Miki from the Keck School of Medicine at the University of Southern California and his colleagues is so important. In this paper, which was published in the journal Stem Cell Research and Therapy (Stem Cell Research & Therapy 2016, 7:8 doi:10.1186/s13287-015-0258-z), Miki and others examined the characteristics of amniotic stem cells grown in xeno-free media (see Mitry RR, Lehec SC, Hughes RD, Methods Mol Biol 2010, 640:107-13; Polchow B, et al. J Transl Med 2012, 10:98; Stacy GN, Masters JR, Nat Protoc 2008, 3:1981-89; Miki T, et al. Curr Protoc Stem Cell Biol 2010, Chapter 1:Unit 1E.3) and compared them to stem cells grown in standard culture media. This is a very important exercise, since amniotic stem cells are easily accessible as source of material for regenerative treatments that can be banked and immunotype matched for clinical applications.

Miki and others isolated human amniotic epithelial cells from newborn babies with the consent of their parents and then stored the cells at −160 °C in one of five commercially available culture media. Miki and his group used cells frozen in standard media containing fetal bovine serum as controls to which all the other cells were compared. Then they thawed the cells, and tested their viability, mitochondrial integrity, and senescence status (tendency to fall asleep in culture and stop growing). They also examined gene expression profiles in these cells by using quantitative real-time PCR. Flow cytometry was used to identify the stem cell surface markers.

The results were encouraging and interesting. There were no significant differences in viability and growth in cells grown and preserved in xeno-free media versus standard cryopreservation medium. Additionally, comparisons of the cells grown in the different cryopreservation media did not reveal significant differences in the senescence status, mitochondria, or overall morphology of the cells. The upshot is that the cells preserved in standard or xeno-free media looked and grew the same after being thawed. There were some differences in the expression of stem cell marker genes (e.g., OCT4, SOX2, and NANOG) and a particular cell surface marker (TRA1-60) following cryopreservation in different xeno-free media. However, it turns out these differences were slight and, overall, not statistically significant. Again, the upshot is that there were small differences in gene expression, but these differences did not amount to a hill of beans.

Miki and his colleagues have nicely shown that cryopreserving amnion-derived stem cells in xeno-free media is feasible and does not affect the characteristics of the cells. This paper also suggests that such xeno-free media can be used to establish a bio-bank of human amnion-derived stem cells for future clinical application.

Well that’s one hurdle vaulted. Now Miki and others need to figure out which of the xeno-free media is the best and optimize that medium to for improved preservation of stem cell-like characteristics in these cells.

Making Cartilage from Umbilical Cord Stem Cells Without Growth Factors

Loïc Reppel and his colleagues at CNRS-Université de Lorraine in France have found that mesenchymal stem cells from human umbilical cord can not only be induced to make cartilage, but that these remarkable cells can make cartilage without the use of exogenous growth factors.

Mesenchymal stromal/stem cells from bone marrow (BM-MSC) have, for some time, been the “all stars” for cartilage regeneration. In fact, a very innovative clinic near Denver, CO has pioneered the use of BM-MSCs for patients with cartilage injuries. Chris Centeno, the mover and shaker, of this clinic has carefully documented the restoration of articular cartilage in many patients in peer-reviewed articles.

However, there is another “kid’ on the cartilage-regeneration block; mesenchymal stromal/stem cells from Wharton’s jelly (WJ-MSC). The advantages of these cells are their low immunogenicity and large cartilage-making potential. In this paper, which was published in Stem Cell Research and Therapy, Reppel and others evaluated the ability of WJ-MSCs to make cartilage in three-dimensional culture systems.

Reppell and his coworkers embedded WJ-MSCs isolated from the umbilical cords of new-born babies in alginate/hyaluronic acid hydrogel and grew them for over 28 days. These hydrogels were constructed by the spraying method. The hydrogel solution (for those who are interested, it was 1.5 % (m/v) alginate and hyaluronic acid (ratio 4:1) dissolved in 0.9 % NaCl) was sprayed an airbrush connected to a compressor. The solution was seeded with WJ-MSCs and then sprayed on a sterile glass plate. The hydrogel was made solid (gelation) in a CaCl 2 bath (102 mM for 10 minutes). Then small cylinders were cut (5 mm diameter and 2 mm thickness) with a biopsy punch. Then Reppel and others compared the chondrogenic differentiation of WJ-MSC in these three-dimensional scaffolds, without adding growth factors with BM-MSC.

Illustration of protocol steps used to perform scaffold construct and chondrogenic differentiation. After monolayer expansion, MSC were seeded at 3× 10 6 cells/mL of Alg/HA hydrogel. Hydrogel was sprayed, gelated, and cut into 5 mm diameter cylinders; scale bar = 5 mm. Scaffolds were cultivated in a 48-well plate in differentiation medium for 28 days. Alg/HA alginate/hyaluronic acid, MSC mesenchymal stromal/stem cells, P3 passage 3

Illustration of protocol steps used to perform scaffold construct and chondrogenic differentiation. After monolayer expansion, MSC were seeded at 3× 10 6 cells/mL of Alg/HA hydrogel. Hydrogel was sprayed, gelated, and cut into 5 mm diameter cylinders; scale bar = 5 mm. Scaffolds were cultivated in a 48-well plate in differentiation medium for 28 days. Alg/HA alginate/hyaluronic acid, MSC mesenchymal stromal/stem cells, P3 passage 3

After 3 days in culture, WJ-MSCs seemed nicely adapted to their new three-dimensional culture system without any detectable damage. From day 14 – 28, the proportion of WJ-MSC cells that expressed all kinds of cell surface proteins characteristic of MSCs (i.e., CD73, CD90, CD105, and CD166) decreased significantly. This suggests that these cells were differentiating into some other cell type.

After 28 days in this scaffold culture, both WJ-MSCs and BM-MSCs showed strong upregulation of cartilage-specific genes. However, WJ-MSCs exhibited greater type II collagen synthesis than BM-MSCs, and these differences were evident at the RNA and protein levels. Collagen II is a very important molecule when it comes to cartilage synthesis because chondrogenesis, otherwise known as cartilage production, occurs when MSCs differentiate into cartilage-making cells known as chondroblasts that begins secreting aggrecan and collagen type II that form the extracellular matrix that forms cartilage. Unfortunately, in order to complete the run to mature cartilage formation, the chrondrocytes must enlarge (hypertrophy), and express the transcription factor Runx2 and secrete collagen X. Unfortunately, WJ-MSCs expressed Runx2 and type X collagen at lower levels than BM-MSCs in this culture system.

Matrix synthesis detected after 28 days of chondrogenic induction. Proteoglycans and total collagen were stained by Alcian blue and Sirius red (a), respectively. To explore the synthesis of various collagens in depth, immunofluorescence (b) and immunohistochemistry staining (c) were performed and detected using fluorescence microscopy and light microscopy, respectively; scale bar = 100 μm. BM-MSC bone marrow-derived mesenchymal stromal/stem cells, WJ-MSC Wharton’s jelly-derived mesenchymal stromal/stem cells

Matrix synthesis detected after 28 days of chondrogenic induction. Proteoglycans and total collagen were stained by Alcian blue and Sirius red (a), respectively. To explore the synthesis of various collagens in depth, immunofluorescence (b) and immunohistochemistry staining (c) were performed and detected using fluorescence microscopy and light microscopy, respectively; scale bar = 100 μm. BM-MSC bone marrow-derived mesenchymal stromal/stem cells, WJ-MSC Wharton’s jelly-derived mesenchymal stromal/stem cells

These experiments only examined cells in culture, which is not the same as placing cells in a living animal, but it is a start. Thus, when they are seeded in the hydrogel scaffold, WJ-MSCs and BM-MSCs, after 4 weeks, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins in the absence of growth factors. In order to properly make cartilage in clinical applications, WJ-MSCs must go the full way and express high levels of Runx2 and collagen X. However, these experiments show that WJ-MSCs, which in the past were medical waste, are a potential alternative source of stem cells for cartilage tissue engineering.

Reppel and his colleagues note in their paper that to improve cartilage production from WJ-MSCs, it might be important to mimic the physiological environment in which chondrocytes normally find themselves. For example, they could apply mechanical stress or even a low-oxygen culture system. Additionally, Reppel and others could apply stratified cartilage tissue engineering. Reppel thinks that they could adapt their spraying method to design new stratified engineered tissues by applying progressive cells and spraying hydrogel layers one at a time.

All in all, cartilage repair based with WJ-MSC embedded in Alginate/Hyaluronic Acid hydrogel will hopefully be tested in laboratory animals and then, perhaps, if all goes well, in clinical trials.

Pluripotent Stem Cells Used to Make a Functional Thyroid

The thyroid gland sits over the main cartilage of the larynx and produces thyroid hormone (thyroxine); a hormone that regulates the basal metabolic rate. When the thyroid slows down and fails to make sufficient amounts of thyroid hormone, the result is a condition called hypothyroidism. The symptoms of hypothyroidism are fatigue, weakness, weight gain or increased difficulty losing weight, coarse, dry hair, dry, rough pale skin, hair loss, cold intolerance, muscle cramps and frequent muscle aches, constipation, depression, irritability, memory loss, abnormal menstrual cycles and decreased libido.

If someone has any evidence of a thyroid tumor, then the thyroid is removed, and the patient must take oral thyroid hormone. Because getting the dose right can be difficult, we might ask, “Can we replace the thyroid with stem cell treatments?”

Human pluripotent stem cells can differentiate into balls of cells that are mini-organs called “organoids.”  Unfortunately, if left to themselves, the formation of these organoids is rather haphazard and the cells tend to differentiate into a whole host of different cell types. This is not fatal, however, since the differentiation of these stem cells can be orchestrated by using growth factors or certain culture conditions. Can we use such innovations to make a minithyroid?

Darrell Kotton and his group at Boston University School of Medicine Pulmonary, Allergy, Sleep and Critical Care Medicine have spent their time tweaking the conditions to drive human pluripotent stem cells to form thyroid cells. A new study of theirs that appears in the journal Cell Stem Cell details how the use of two growth factors, BMP4 and FGF2 can drive pluripotent stem cells to commit to thyroid cell fates.

In order to make thyroid cells from embryonic stem cells (ESCs), Kotton and his group had to make endodermal progenitors from them first. Fortunately, a study from Kotton’s own laboratory that was published in 2012 employed a technique used in several other papers that grew ESCs in a serum-free medium with a growth factor called activin. Christodoulou, C., and others (J. Clin. Invest. 121, 2313–2325) showed that over 80 percent of the ESCs grown under these conditions differentiated into endodermal progenitors.  When Kotton and his colleagues cultured these endodermal progenitors in BMP4 and FGF2, some of them differentiated into thyroid progenitor cells. Interestingly, this mechanism by which thyroid-specific cell fates are specified is conserved in creatures as disparate as frogs and mice.

To make mature thyroid cells from these progenitors, a three-dimensional culture system was used in combination with thyroid stimulation hormone and dexamethasone. Under these conditions, the cells formed spherical thyroid follicles that secreted thyroid hormone. To perfect their protocols, Kotton’s group used mouse ESCs, but they additionally showed that this same strategy can make mature thyroid cells from human induced pluripotent stem cells (iPSCs).

Thyroid specification

The appearance of cells in a culture system that look like mature thyroid follicles and express many of the same iodine-metabolizing enzymes as mature thyroid cells is exciting, but can such cells stand in for thyroid tissue in an animal that lacks sufficient thyroid tissue?

Kotton’s laboratory took this to the next step by transplanting their cultured thyroid follicles into laboratory mice that lacked a functional thyroid. These transplants were not inserted into the neck of the animal, but instead were place underneath the kidney, which is area rich in blood vessels. Interestingly, the implanted thyroid “organoids” or little organs did not fall apart upon transplantation. Instead they retained their characteristic structure. More interestingly, these organoids kept expressing iodine-metabolizing enzymes and made thyroid hormone. The synthesis and release of thyroid hormone was also regulated by the hypothalamic hormone thyroid stimulating hormone (TSH). TSH is made and released in response to insufficient thyroid hormone levels. The thyroid responds to TSH by making a releasing more thyroid hormone, which causes a feed-back inhibition of the release of TSH. The fact that these implanted organoids were properly regulated by TSH bespeaks of the maturity of these cells. Also, significantly, none of the laboratory animals showed any signs that the implanted cells had formed any tumors.

Kotton and his coworkers were also able to used human ESCs and human iPSCs to make thyroid organoids. Human iPSCs-derived thyroid organoids were made from human patients with normal thyroid function and from hypothyroid children who carry a loss-of-function mutation in the NKX2-1 gene. This show that Kotton’s system can be used as a model to study inherited thyroid deficiencies. However, there is even more excitement that this system or something similar to it might be useful to safely treat thyroid loss in patients who have lost their thyroid as a result of cancer, or injury.

Small Molecule Supercharges Human Cardiac Stem Cells

HO-1 or heme oxygenase is an enzyme that degrades heme groups to biliverdin, iron, and carbon monoxide. It is induced in cells in response to oxidative stress. Overexpression of HO-1 can make cells more resistant to oxidative stress. The highest levels of HO-1 are found in the spleen, where old red blood cells are sequestrated and destroyed.

Mesenchymal stem cells (MSCs) from bone marrow have been genetically engineered to overexpress HO-1 survive much better when implanted into the hearts of animals that have recently suffered a heart attack (Zeng B, et Al, Biomed Sci. 2010 Oct 7;17:80; Yang JJ et al Tohoku J Exp Med. 2012;226(3):231-41). Such cells also increase the density of blood vessels in infarcted tissue, and HO-1 has been postulated to increase blood vessel production (Jang YB et al Chin Med J (Engl). 2011 Feb;124(3):401-7).

These previous experiments show that HO-1 can increase the survival and therapeutic abilities of MSCs. Can increasing the levels of HO-1 do the same for other types of stem cells?

Stuart Atkinson at the Stem Cell Portal web site has highlighted a new paper that was published in the journal Stem Cells that has examined increasing the levels of HO-1 in Cardiac Stem Cells (CSCs).

CSCs are a resident stem cell in the heart that can be isolated from heart patients during heart surgeries. Animal studies and clinical trials have shown that implantation of CSCs soon after a heart attack can produce significant increases in heart function (Bearzi C, et al. Proc Natl Acad Sci U S A 2007;104:14068-14073; Bolli R, et al Lancet. 2011 Nov 26;378(9806):1847-57). Unfortunately, the success of this clinical has been called into questioned by some problems with the data reported in this paper. However, animal studies suggest that the effectiveness of CSCs is compromised by their limited ability to survive in the heart after a heart attack (Hong KU, et al. PLoS One 2014;9:e96725). Therefore, increasing the survival of CSCs might increase their therapeutic efficacy.

Atkinson notes that the compound cobalt protoporphyrin (CoPP) can induce the expression of higher levels of HO-1 and thereby increase the resistance of the cells to oxidative stress and augment cell survival. Therefore, Robert Bolli from the University of Louisville, Kentucky and his colleagues, in collaboration with researchers from the Albany Medical College have treated CSCs with CoPP and these tested their ability to heal the heart after a heart attack.

Bolli and others isolated human CSCs from patients undergoing CABG (cardiac artery bypass graft) surgery, and grew them in culture to beef up the numbers of cells. After a short time in culture, the CSCs were incubated with CoPP for 12 hours. Then Bolli and his team transplanted these human CSCs that were also labeled with green fluorescent protein (GFP) into the hearts of mice that had suffered rather massive heart attacks and had undergone 35 days of reperfusion. The GFP allowed Bolli and others to detect the presence of the implanted CSCs in the rodent heart tissue.

When these hearts of these mice were examined one and five weeks after CSC transplantation, the CoPP-treated CSCs showed substantially higher levels of survival in the mouse hearts. The other two groups of mice included those transplanted with non-pretreated CSCs, and mice treated with the culture medium used to grow the CSCs, and the pretreated CSCs survival significantly better than the non-pretreated CSCs.

CoPP pretreatment seems to augment cell survival, but do the surviving cells increase heart function? Bolli and others used echocardiogram to measure heart function, and echocardiographic assessment 5 weeks after CSC transplantation showed that the CoPP-preconditioned CSCs elicited greater improvement in remodeling of the left ventricle. Additionally, the hearts of the animals that received CoPP-pretreated CSCs showed improved movement of the walls of the heart during its pumping cycle, and better overall performance of the heart in general. Both pretreated and the non-pretreated CSCs, but not CSC culture growth medium shrank the amount of scar tissue in the heart and grew new heart tissue. However, The CoPP-pretreated CSCs were obviously superior to the non-pretreated CSCs at increasing the mass of heart muscle (see here for pictures).

These experiments might very well unravel a burning controversy surrounding CSCs. Bolli’s experiment show that can definitely grow new heart muscle. However, the bulk of the experiments with CSCs strongly suggest that these cells improve heart function by secreting pro-healing molecules without directly contributing to the regrowth of heart muscle. These papers probably observed the effects of CSCs that were transplanted into the heart, but did not survive very long. Bolli and his colleagues, on the other hand, were able to implant CSCs and survived for a much longer time in the hearts. Incidentally, Bolli and his team showed that the implanted CSCs expressed heart muscle-specific genes, which corroborated that these cells were differentiating into heart muscle cells, even though the proportion of cells that formed new heart muscle was relatively small.

In summary, CoPP pretreatment of cell seems to be feasible, safe, and effective as a means to improve CSC-based therapy. Even though It is likely that paracrine mechanisms are essential for CSC-based healing, the ability of CSCs to differentiate into heart muscle cells also seems to be an essential part of the means by which CSCs heal the heart after a heart attack. Thus more work is certainly warranted, but this is a fine start to what might be a simple, but effective way to increase the effectiveness of our own CSCs.

How Stem Cell Therapy Protects Bone In Lupus

Systemic Lupus Erythematosis, otherwise known as lupus, is an autoimmune disease cause your own immune system attacking various cells and tissues in your body. Lupus patients can suffer from fatigue, joint pain and selling and show a marked increased risk or osteoporosis.

Clinical trials have established that infusions of mesenchymal stem cells (MSCs) can significantly improve the condition of lupus patients, but exactly why these cells help these patients is not completely clear. Certainly suppression of inflammation is probably part of the mechanism by which these cells help lupus patients, but how do these cells improve the bone health of lupus patients?

Songtao Shi and his team at the University of Pennsylvania have used an animal model of lupus to investigate this very question. In their hands, transplanted MSCs improve the function of bone marrow stem cells by providing a source of the FAS protein. FAS stimulates bone marrow stem cell function by means of a multi-step, epigenetic mechanism.

This work by Shi and his colleagues has implications for other cell-based treatment strategies for not only lupus, but other diseases as well.

“When we used transplanted stem cells for these diseases, we didn’t know exactly what they were doing, but saw that they were effective,” said Shi. “Now we’ve seen in a model of lupus that bone-forming mesenchymal stem cell function was rescued by a mechanism that was totally unexpected.”

In earlier work, Shi and his group showed that mesenchymal stem cell infusions can be used to treat various autoimmune diseases in particular animals models. While these were certainly highly desirable results, no one could fully understand why these cells worked as well as they did. Shi began to suspect that some sort of epigenetic mechanism was at work since the infused MSCs seemed to permanently recalibrate the gene expression patterns in cells.

In order to test this possibility, Shi and others found that lupus mice had a malfunctioning FAS protein that prevented their bone marrow MSCs from releasing pro-bone molecules that are integral for bone maintenance and deposition.

A deficiency for the FAS protein prevents bone marrow stem cells from releasing a microRNA called miR-29b.  The failure to release miR-29b causes its concentrations to increase inside the cells.  miR-29b can down-regulate an enzyme called DNA methyltransferase 1 (Dnmt1), and the buildup of miR-29b inhibits Dnmt1, which causes decreased methylation of the Notch1 promoter and activation of Notch signaling.  Methylation of the promoters of genes tends to shut down gene expression, and the lack of methylation of the Notch promoter increases Notch gene expression, activating Notch signaling.  Unfortunately, increased Notch signaling impaired the differentiation of bone marrow stem cells into bone-making cells.  Transplantation of MSCs brings FAS protein to the bone marrow stem cells by means of exosomes secreted by the MSCs.  The FAS protein in the MSC-provided exosomes reduce intracellular levels of miR-29b, which leads to higher levels of Dnmt1.  Dnmt1 methylates the Notch1 promoter, thus shutting down the expression of the Notch gene, and restoring bone-specific differentiation.

Shi and others are presently investigating if this FAS-dependent process is also at work in other autoimmune diseases.  If so, then stem cell treatments might convey similar bone-specific benefits.

Faster Bone Regeneration With a Little Wnt

Nick Evans and his colleagues at the University of Southampton, UK have discovered that transient stimulation of the Wnt signaling pathway in bone marrow stem cells expands them and enhances their bone-making ability. This finding has led to an intense search for drugs that can stimulate the Wnt pathway in order to stimulate bone formation in wounded patients.

The Wnt pathway is a highly conserved pathway found in sponges, starfish, sharks, and people. Wnt signaling controls pattern formation during development, and the growth of stem cells during healing.

When it comes to healing, bone fractures represent a sizeable societal problem, particularly among the aged. While most fractures heal on their own, approximately 10 percent of all fractures take over six months to heal or never heal at all. In the worse cases, fracture patients can require several surgeries or might need amputation in desperate cases.

According the Evans, he and his research group are screening a wide range of chemicals to determine if they stimulate Wnt signaling. If such chemicals prove safe to use in laboratory animals, then they might become clinical tools to help stimulate bone formation and healing in patients with recalcitrant fractures.

Research from Evans’ group has shown that transient stimulation of the Wnt signaling pathway in isolated bone marrow cells increases the number of bone-making progenitor cells. However, if the Wnt pathway is activated for too long a time period, this regenerative effect is lost or even reversed. Hence the need to develop treatments that deliver small molecules that stimulate Wnt signaling in bone marrow cells for a specified period of time and in a targeted fashion.

Evans and his group have used nanoparticles loaded with Wnt proteins to do exactly that. The feasibility of this technology and its effectiveness requires further work, but the promise is there and the idea is more than a little intriguing.