Regenerating Whole Teeth With A Tissue-Engineered Scaffold

As tissues go, teeth are relatively simple. They only consist of a few cell types, arranged in a rather straight-forward manner. Therefore, regenerating teeth, while more difficult than it seems, should represent a tractable problem for stem cell biologists and tissue engineers. While some progress has been made, tooth regeneration procedures will require more fine-tuning before they will be hailed as successful.

Tzong-Fu Kuo and others from the School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan have examined the feasibility of whole-tooth regeneration in minipigs. Kuo and his group used a tissue-engineered tooth germ-like construct.

To construct their tooth germ constructs, Kuo and his colleagues extracted dental pulp from upper incisors, canines, premolars, and molars from mature miniature pigs. They grew the dental pulp tissue in culture in order to expand the faster-growing dental pulp stem cells (DPSCs) that can outgrowth everything else from the pulp in culture. They differentiated the DPSCs into odontoblasts, which make the dentine of the tooth, and osteoblasts, which make bone. Kuo’s team also acquired gingival epithelial cells from the gums of the minipigs.

Next the gum epithelial cells, odontoblasts, and osteoblasts were implanted onto the surface (upper, and lower layers, respectively), of a bioactive scaffold. This scaffold had the odontoblasts inside, the osteoblasts outside, and the gum epithelial cells outside the osteoblasts. Then Kuo and his coworkers transplanted these seeded bioactive scaffolds into the tooth sockets of the lower jaw of a minipig whose lower first and second molar tooth germs were removed.

13.5 months after the scaffolds were implanted, seven of eight pigs had formed two new teeth that had crowns, roots, and pulp. When the newly-formed teeth were extracted and sectioned, they had enamel-like tissues, dentin, cementum, odontoblasts, and periodontal tissues.

A fascinating finding in this study was that all the pigs, without exception, had regenerated molar teeth regardless of the original tooth from which the DPSCs were isolated. As an important control, minipigs that had their tooth germs removed or received empty scaffolds did not develop teeth.

This study from Kuo’s laboratory showed that implantation of a tooth germ-like structure can produce a complete tooth can do so successfully and efficiently. This study also established that the location of the implant seemed to deeply influence the morphology of the regenerated tooth.

Stem-Cell Dental Implants Grow New Teeth in Your Mouth

Dr. Jeremy Mao is the Edward V. Zegarelli Professor of Dental Medicine at Columbia University Medical Center. Mao and his colleagues have published a novel technology that includes a growth factor-infused, three-dimensional scaffold that has the potential to regenerate an anatomically correct tooth in the mouth just nine weeks after implantation. By this procedure, which was developed in the university’s Tissue Engineering and Regenerative Medicine Laboratory, Mao can direct the body’s own stem cells to migrate to the scaffold and infiltrate it. Once these stem cells have colonized the scaffold, they will produce a tooth that can grow in the socket and merge with the surrounding tissue and integrate into it.

Tooth scaffold that is completely composed of natural materials.
Tooth scaffold that is completely composed of natural materials.

Mao’s technique not only eliminates the need to grow teeth in a culture, but it can regenerate anatomically correct teeth by using the body’s own resources. If you factor in the faster recovery time and the comparatively natural process of regrowth (as opposed to implantation), you have a massively appealing dental treatment.

Columbia University has already filed patent applications in regard to this technology. They are also seeking associates to aid in its commercialization. Mao is also considering the best approach for applying his technique to cost-effective clinical therapies.

The Isolation of Dental Stem Cell Lines and How They Repair Teeth

Research teams at INSERM and Paris Descartes University have isolated dental stem cell lines and detailed the natural mechanism by which such cells repair lesions in teeth. This discovery could provide the foundation for therapeutic strategies that mobilize resident dental stem cells and amplify their intrinsic capacity for repair.

Teeth are mineralized organs that are anchored within the gums by roots. The outermost layer of the tooth is the enamel, which is one of the hardest substances in the body. Beneath the enamel is the dentine (sometimes known as the ivory), and the dentin (dentine if you are British) is filled with microscopic tubes that radiate from the inner pulp to the surface. These dentinal tubules contain cells called odontoblasts, which are the cells that made the dentin in the first place, and dentinal fluid (contains a mixture of albumin, transferrin, tenascin and proteoglycans). The odontoblasts maintain the dentin, but as we age, the dentin tubules calcify. Dentin is a yellowish, bone-like matrix that is porous. It is softer than enamel and decays more rapidly and is subject to severe cavities if not properly treated. Beneath the dentin is the pulp, which contains blood vessels and nerves and also houses a resident stem cell population.

Tooth Anatomy

When a dental lesion appears, the dormant stem cells in the pulp awaken and try to repair the tooth, but the means by which these cells do this is unknown.

To address these gaps in our knowledge, researchers from INSERM (French Institute of Health and Medical Research or Institut national de la santé et de la recherche), and the Paris Descartes University have extracted and isolated dental stem cells from the pulp of mouse molars and analyzed them further.

In the midst of their characterization of these cells, the French teams discovered that these dental pulp stem cells possess five different cell surface receptors for the neurotransmitters dopamine and serotonin. The present of these receptors suggested that the response of dental pulp stem cells to tooth injury was mediated by these neurotransmitters. Blood platelets, for example, are activated by binding serotonin and dopamine. Could these dental be activated by similar means?

The first set of experiments examined tooth repair in mice that lacked platelets that produced serotonin or dopamine. Such mice failed to repair tooth lesions, suggesting that serotonin and dopamine are important to inducing stem cell-mediated tooth repair.

Next, these laboratories characterized these five receptors and found that four of them were intimately involved in tooth repair; knocking out any one of the would abrogate tooth repair responses.

“In stem cell research, it is unusual to be simultaneously able to isolate cell lines, identify the markers that allow them to be recognized (here the receptors), discover the signal that recruits them (serotonin and dopamine), and discover the source of that signal (blood platelets). In this work, we have been able, unexpectedly, to explore the entire mechanism,” said Odile Kellermann, the principal author of this work.

Dentists use pulp capping materials like calcium hydroxide and tricalcium phosphate-based biomaterials to repair the tooth and fill lesions. These new findings, however, could produce new therapeutic strategies aimed at mobilizing the resident dental pulp stem cells to magnify the natural reparative capacity of teeth without the use of replacement materials.

Healing Corneal Blindness with Stem Cells from Extracted Teeth

Scientists at the University of Pittsburgh have found a new way to treat corneal blindness, which affects millions of people around the world.

James Funderburgh and his colleagues at the University of Pittsburgh School of Medicine showed that stem cells isolated from the dental pulp of extracted wisdom teeth can be differentiated into specialized cells that can maintain the health and integrity of corneas and rid them of the scars caused by illness or injury that compromise the ability to see clearly.  These cells can be safely injected into the corneas of mice.

According to Funderburgh, who is a professor of ophthalmology, this new approach can replace damaged corneal eye tissue with tissue made from the patient’s own cells rather than cells from a donor. Such a procedure circumvents the problems of immunological rejection that dog the reconstruction of corneal tissue with cells from donors. Furthermore, donor corneas are in short supply in certain parts of the world (e.g., Africa and Asia).

“Our work is promising because using the patient’s own cells for treatment could help avoid these problems,” said Dr. Funderburgh, who is the senior author of a new paper describing the research, in a written statement.

A post-doctoral research fellow in Dr. Funderburgh’s laboratory, Dr. Fatima Syed-Picard, took cells from the pulp of extracted wisdom teeth and chemically processed them to differentiate them into specialized corneal cells. Then Syed-Picard and others injected these “keratocytes” into the corneas of healthy mice. Once in the eyes of laboratory mice, the tooth pulp-derived cells integrated with the existing tissue with no sign of rejection even after several weeks.

Could such a treatment work in human patients? “We are thinking that in the future people may ‘bank’ their extracted wisdom teeth or the cells from those teeth,” Funderburgh told The Huffington Post in an email. “For someone who did not do that it is possible to extract dental pulp with a root canal procedure, but this is still hypothetical. In the worst-case scenario, someone might consider having a tooth extracted to provide cells for this procedure.”

Last year more than 70,000 corneal transplants were performed in the U.S., According to Kevin Corcoran, president and CEO of the Washington, D.C.-based Eye Bank Association of America (EBAA), there were more than 70,000 corneal transplants performed in the US alone.

“There’s a lot of exciting research being done in the area of [corneal] transplant, and EBAA is interested in any outcome that can help restore sight to the blind or visually impaired,” said Corcoran, who was unfamiliar with the Pitt research.

Dr. Syed-Picard stressed that this research is still in the formative stages and the results are preliminary, and added that it would probably take a few years before human testing could begin. The next step, she said, would be to conduct a similar set of experiments in rabbits.

Bone Marrow Stem Cells and Tissue Engineering Give a Woman a New Smile

Massive injuries to the face can cause bone loss and “tooth avulsion.” Medically speaking, avulsion simply refers to the detachment of a body structure from its normal location by means of surgery or trauma. Dental implants and help with lost teeth, but if the facial bone has suffered so much loss that you cannot place implants in them, then you are out of luck. Dental prostheses can help, but these do not always fit very well.

Darnell Kaigler and his group at the University of Michigan Center for Oral Health wanted to help a 45-year-old woman who had lost seven teeth and a good portion of her upper jaw bone (maxilla) as a result of massive trauma to the face. This poor lady had some dentures that did not fit well and a mouth that did not work well.

Bone can be grown from stem cells, but getting those stem cells to survive and do what you want them to do is the challenge of regenerative medicine. Therefore, Dr. Kraigler and his group used a new technique to help this young lady, and their results are reported in the December 2014 issue of the journal Stem Cells Translational Medicine.

First, Kraigler and his co-workers extracted bone marrow stem cells from a bone marrow aspiration that was taken from the upper part of the hip bone (the posterior crest of the ilium for those who are interested).  They used a product called ixmyelocel-T from Aastrom Biosciences in Ann Arbor , MI. This product is a patient-specific, expanded multicellular therapy, cell-processing system that selectively expands mesenchymal cells, monocytes and alternatively activated macrophages, up to several hundred times more than the number found in the patient’s bone marrow, while retaining many of the hematopoietic cells collected from only a small sample (50ml) of the patient’s bone marrow. Thus, the healing cells from the bone marrow are grown and made healthy, after which the cells were bagged and frozen for later use.


Then the patient was readied for the procedure by having the gum tissue cut and lifted as a flap of tissue (under anesthesia, or course). Then four holes were drilled into the bone and setting screws were inserted. This is an important procedure, because implanted stem cells will not survive unless they have blood vessels that can bring them oxygen and nutrients. By drilling these holes, the tissue responds by making new blood vessels. To this exposed surface, the bone marrow-derived stem cells were applied with a tricalcium phosphate (TCP). TCP is a salt that will induce mesenchymal stem cells to form bone. Once the TCP + stem cell mixture was applied to the gum, a collagen membrane was placed over it, and the gum was then sewn shut with sutures.

Cell transplantation procedure. Front view (A) and top view (B) of the initial clinical presentation showing severe hard and soft tissue alveolar ridge defects of the upper jaw. Following elevation of a full-thickness gingival flap, the images show front view (C) and top view (D) of the severely deficient alveolar ridge, clinically measuring a width of only 2–4 mm. Front view (E) and top view (F) of the placement of “tenting” screws in preparation of the bony site to receive the graft. Placement of the β-tricalcium phosphate (seeded with the cells 30 minutes prior to placement at room temperature) into the defect (G), with additional application of the cell suspension following placement of the graft in the recipient site (H). Placement of a resorbable barrier membrane (I) to stabilize and contain the graft within the recipient site, and top view (J) of primary closure of the flap.
Cell transplantation procedure. Front view (A) and top view (B) of the initial clinical presentation showing severe hard and soft tissue alveolar ridge defects of the upper jaw. Following elevation of a full-thickness gingival flap, the images show front view (C) and top view (D) of the severely deficient alveolar ridge, clinically measuring a width of only 2–4 mm. Front view (E) and top view (F) of the placement of “tenting” screws in preparation of the bony site to receive the graft. Placement of the β-tricalcium phosphate (seeded with the cells 30 minutes prior to placement at room temperature) into the defect (G), with additional application of the cell suspension following placement of the graft in the recipient site (H). Placement of a resorbable barrier membrane (I) to stabilize and contain the graft within the recipient site, and top view (J) of primary closure of the flap.

Four months later, the patient underwent a cone-beam computed tomography (CBCT) scan. The bone regrowth can be seen in the figure below.

Cone-beam computed tomography (CBCT) scans. CBCT scans were used to render three-dimensional reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points. (A, B): The initial clinical presentation shows 75% jawbone width deficiency. (C, D): Immediately following cell therapy grafting, there is full restoration of jawbone width. (E, F): Images show 25% resorption of graft at 4 months and overall net 80% regeneration of the original ridge-width deficiency.
Cone-beam computed tomography (CBCT) scans. CBCT scans were used to render three-dimensional reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points. (A, B): The initial clinical presentation shows 75% jawbone width deficiency. (C, D): Immediately following cell therapy grafting, there is full restoration of jawbone width. (E, F): Images show 25% resorption of graft at 4 months and overall net 80% regeneration of the original ridge-width deficiency.

According to the paper, there was an “80% regeneration of the original jawbone.”

Into this newly regenerated bone, permanent dental implants were placed. The results are shown below.

Complete oral rehabilitation. Clinical presentation of the patient prior to initiation of treatment (A) and following completed oral reconstruction (B). (C): Periapical radiographs of oral implants showing osseointegration of implants and stable bone levels at the time of placement, 6 months following placement, and 6 months following functional restoration and biomechanical loading of implants with a dental prosthesis.
Complete oral rehabilitation. Clinical presentation of the patient prior to initiation of treatment (A) and following completed oral reconstruction (B). (C): Periapical radiographs of oral implants showing osseointegration of implants and stable bone levels at the time of placement, 6 months following placement, and 6 months following functional restoration and biomechanical loading of implants with a dental prosthesis.

Pardon me, but permit me an unprofessional moment when I say that this is really cool.  Of course, this patient will need to be observed over the next several years to determine the longevity of her bone regeneration, but the initial result is certainly something to be excited about.

Tricalcium phosphate or TCP has been used to induce the bone-making activities of mesenchymal stem cells.  It has also been used in several animal studies as a delivery vehicle for mesenchymal stem cells (for example, see Rai B, et al., Biomaterials 2010, 31:79607970; Krebsbach PH, et al., Transplantation 1997, 63:10591069; Zhou J, et al., Biomaterials 2010, 31:11711179).  TCP also seems to support stem cell proliferation, survival, and differentiation into bone.  Kresbach and others showed that TCP most consistently yielded bone formation when used as a delivery vehicle for mesenchymal stem cells compared to other biomaterials commonly used, such as gelatin sponges and demineralized bone matrix.  However, there are no studies that have ascertained how well stem cells attach to TCP, and this attachment is an important factor in determining how many stem cells reach the site of injury.  This study by Kaigler and his group (A. Rajan and others) showed that a 30-minute incubation of the cells with TCP gave sufficient attachment of the cells to the TCP for clinical use.  The efficiency of this incubation period was also not affected by the temperature.  

The other exciting features of this paper, is that most of the materials used in this study were commercially available.  The bone marrow stem cell isolation technique was pioneered by Dennis JE, and others in their 2007 article in the journal Stem Cells (25:25752582).  Effective commercialization of this technique has shown the efficacy of this procedure for clinical use.  This paper also shows the clinical feasibility of using TCP as a delivery vehicle for mesenchymal stem cell-based bone treatments.

In conclusion, I will quote the authors: “Cell survival and seeding efficiency in the context of tissue engineering and cell-therapy strategies are critical parameters for success that have not been rigorously examined in a clinical context. This study defined optimized conditions for these parameters using an autologous stem cell therapy to successfully treat a patient who had a debilitating craniofacial traumatic deficiency. To our knowledge, there have been no other clinical reports of cell therapy for the treatment of craniofacial trauma defects. This clinical report serves as solid foundation on which to develop more expanded studies using this approach for the treatment of larger numbers of patients with other debilitating conditions (e.g., congenital disorders) to further evaluate efficacy and feasibility.”

Regenerating Tooth Roots With Biomaterials

Several different types of stem cells can regenerate tooth enamel, but regenerating the tooth root has proven quite difficult.


As you can see from the image above, the tooth root is covered with a tough, fibrous covering called the cementum.  The cementum connects the tooth root to the alveolar bone of the upper and low jaw by means of the periodontal membrane.  the cementum is a thin layer of bone-like material that covers the roots.  It is yellowish and softer than either dentine or enamel.  It is made by a layer of cementum-producing cells called cementoblasts that are adjacent to the dentine.  The periodontal ligament is cellular and its fibers hold the tooth in its socket, which are embedded in the cementum, as shown in the micrograph below.  The complexity of this structure shows you why regenerating this structure is so difficult.

Cementum-peridontal ligament

Howwever, a new study from the laboratory of Weihua Guo at Sichuan University, China has shown that platelet-rich fibrin (PRF) and treated dentin matrix (TDM) can concentrate a variety of various growth factors that summon native stem cells to them, and induce them to regenerate the tooth root.

Guo’s laboratory examined the ability of PRF and TDM to summon endogenous stem cells to the site of an extracted tooth in order to initiate regeneration of the tooth root.  Tooth roots contain soft and hard periodontal tissues, and if periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMSCs) could be recruited to the site of tooth extraction by PRF and TDM, then maybe they could initiate tooth root regeneration.

Beagles were used as a transplantation model for this experiment.  After tooth extraction PRF and TDM implants were embedded in the tooth socket.  Also, these matrices were examined in cell culture with  PDLSCs and BMSCs.

PRF significantly recruited and stimulated the growth of both PDLSCs and BMSCs in culture.  In combination, PRF and TDM induced cell differentiation of these implanted stem cell populations.  PRF and TDM induced the expression of mineralization-related genes, such as bone sialoprotein (BSP) and osteopotin (OPN) after only one week in culture.

When implanted into the tooth sockets of beagles that had teeth extracted, transplantation platelet-rich fibrin made from the dog’s own blood products, and TDM made from other animals into fresh tooth extraction socket successfully regenerated the tooth root 3 months after the surgery.  The cementum and periodontal ligament (PDL)-like tissues with properly orientated fibers were clearly present, and the presence of these structures is indicative of functional restoration.

These results suggest that tooth root and the connection of the tooth root to the alveolar bone by cementum and peridontal ligaments can be effectively regenerated through the implantation of PRF and TDM in a tooth socket.  It seems to achieve this regeneration by summoning BMSCs and PDLSCs.  These cues provided by these matrices and the microenvironment provided by the tooth socket are key factors for this regeneration. This strategy provides a genuine clinical pathway toward tooth root regeneration in human patients with destroying human embryos.

Gum Nerve Cells Become Tooth-Specific Mesenchymal Stem Cells

Stem cells self-renew and also produce progeny that differentiate into more mature cell types. The neurons and glia that compose nervous systems are examples of mature cells and these cells can be produced from embryonic stem cells, induced pluripotent stem cells, or neural stem cells. However, the reverse does not occur during development; more mature cells do not de-differentiate into less mature cells types. Development tends to be a one-direction event.

However, researchers have now discovered that inside teeth, nervous system cells can transform back into stem cells. This unexpected source of stem cells potentially offers stem cell scientists a new starting point from which to grow human tissues for therapeutic or research purposes without using embryos.

“More than just applications within dentistry, this finding can have very broad implications,” says developmental biologist Igor Adameyko of the Karolinska Institute in Stockholm, who led this new work. “These stem cells could be used for regenerating cartilage and bone as well.”

The soft “tooth pulp” in the center of teeth has been known to contain a small population of tooth-specific mesenchymal stem cells, which can typically differentiate into tooth-specific structures, bones, and cartilage. However, no one has conclusively determined where these stem cells came from. Adameyko hypothesized that if he could trace their developmental lineage, he should be able to recapitulate their development in the laboratory. This might offer new ways of growing stem cells for tissue regeneration.

Adameyko and his and colleagues had already studied glial cells, which are nervous system cells that surround neurons and support them. Several of the nerves that wind through the mouth and gums help transmit pain signals from the teeth to the brain are associated with glial cells.

Adameyko and others used fluorescent labels to mark the glial cells in the gum. When the gum-specific glial cells were observed over time, some of these cells migrated away from neurons in the gums into teeth, where they differentiated into mesenchymal stem cells. These same cells then matured into tooth cells. This work was reported in the journal Nature.

a–c, Incisor traced for 3 days from adult PLP-CreERT2/R26YFP mouse. Note protein gene product 9.5 (PGP9.5)+ nerve fibres (a). b, c, Magnified areas from a. d, e, Incisor traced for 30 days from adult PLP-CreERT2/R26YFP mouse. Note collagen IV+ blood vessels (d). e, YFP+ odontoblasts and adjacent pulp cells. f, Incisor traced for 30 days from Sox10-CreERT2/R26YFP mouse. g–k, Incisor traced for 40 days from PLP-CreERT2/R26Confetti incisor. h–j, Magnified areas from g. Arrow in h indicates a cluster of odontoblasts; arrow in j points at CFP+ and RFP+ cells in proximity to a cervical loop at the base of CFP+ and RFP+ streams shown in g and i. k, Streams of CFP+ and RFP+ pulp cells next to i and j. l, m, Incisor traced for 40 days from PLP-CreERT2/R26Confetti mouse with YFP+ and RFP+ pulp cells adjacent to clusters of odontoblasts with corresponding colours. m, Magnified region from l. n, Stream of pulp cells (arrows) in proximity to the cervical loop; yellow and red isosurfaces mark YFP+ and RFP+ cells. o, p, Progenies of individual MSCs intermingle with neighbouring clones in pulp (o) and odontoblast layer (p), projections of confocal stacks. q, r, Clonal organization of mesenchymal compartment in adult incisor. a–n, Dotted line, enamel organ and mineralized matrix. Scale bars, 100 µm (a, d, f, g, k, l); 50 µm (b, c, e, m–p). CL1 and CL2 indicate labial and lingual aspects of cervical loop. d.p.i., days post-injection. s, Incidence of mesenchymal clones depending on fraction of odontoblasts within the clone. t–v, Proximity of dental MSCs (dMSCs) to cervical loop (CL) correlates with clonal size and proportion of odontoblasts in clone.
a–c, Incisor traced for 3 days from adult PLP-CreERT2/R26YFP mouse. Note protein gene product 9.5 (PGP9.5)+ nerve fibres (a). b, c, Magnified areas from a. d, e, Incisor traced for 30 days from adult PLP-CreERT2/R26YFP mouse. Note collagen IV+ blood vessels (d). e, YFP+ odontoblasts and adjacent pulp cells. f, Incisor traced for 30 days from Sox10-CreERT2/R26YFP mouse. g–k, Incisor traced for 40 days from PLP-CreERT2/R26Confetti incisor. h–j, Magnified areas from g. Arrow in h indicates a cluster of odontoblasts; arrow in j points at CFP+ and RFP+ cells in proximity to a cervical loop at the base of CFP+ and RFP+ streams shown in g and i. k, Streams of CFP+ and RFP+ pulp cells next to i and j. l, m, Incisor traced for 40 days from PLP-CreERT2/R26Confetti mouse with YFP+ and RFP+ pulp cells adjacent to clusters of odontoblasts with corresponding colours. m, Magnified region from l. n, Stream of pulp cells (arrows) in proximity to the cervical loop; yellow and red isosurfaces mark YFP+ and RFP+ cells. o, p, Progenies of individual MSCs intermingle with neighbouring clones in pulp (o) and odontoblast layer (p), projections of confocal stacks. q, r, Clonal organization of mesenchymal compartment in adult incisor. a–n, Dotted line, enamel organ and mineralized matrix. Scale bars, 100 µm (a, d, f, g, k, l); 50 µm (b, c, e, m–p). CL1 and CL2 indicate labial and lingual aspects of cervical loop. d.p.i., days post-injection. s, Incidence of mesenchymal clones depending on fraction of odontoblasts within the clone. t–v, Proximity of dental MSCs (dMSCs) to cervical loop (CL) correlates with clonal size and proportion of odontoblasts in clone.

Before this experiment, it was generally believed that nervous system cells were unable to de-differentiate or revert back to a flexible stem cell state. Therefore, Adameyko said that it was very surprising to see such a process in action. He continued: “Many people in the community were convinced … that one cell type couldn’t switch to the other. But what we found is that the glial cells still very much maintain the capacity” to become stem cells. If stem cell researchers and physicians could master those chemical cues in the teeth pulp that signals glial cells to transform into mesenchymal stem cells, they could generate a new way to grow and make stem cells in the lab.

“This is really exciting because it contradicts what the field had thought in terms of the origin of mesenchymal stem cells,” says developmental biologist Ophir Klein of the University of California, San Francisco, who was not involved in the new work. But it’s also just the first step in understanding the interplay between the different cell populations in the body, he adds. “Before we really put the nail in the coffin in terms of where mesenchymal stem cells are from, it’s important to confirm these findings with other techniques.” If that confirmation comes, a new source of stem cells for researchers will be invaluable, he says.

Laser-Activation of Dental Stem Cells Spurs Dentine Regeneration

A variety of experiments, clinical trials, and strategies have attempted to exploit stem cells as therapeutic agents in regenerative medicine. However, once stem cells are removed from their niches within the body and grown in artificial culture systems their properties can change. Such culture-acquired changes can often compromise the therapeutic potential of some stem cells. For this reason, the development of relatively simple but effective stem cell isolation and manipulation techniques represents someone of the prominent technical hurdles to the clinical use of stem cells.

Several laboratories have used exogenous factors to direct the differentiation of tissue-resident stem cells, but these exogenous factors can often cause unwanted side effects. For this reason, simpler manipulation techniques are always a welcome addition to the armamentarium of stem cell scientists.

To that end, Ashok B. Kulkarni from the National Institute of Dental and Craniofacial Research in Bethesda, MD and David J. Mooney from the Harvard School of Engineering and their colleagues and co-workers have used non-ionizing, low-power laser (LPL) treatments to activate host stem cells and promote tissue regeneration. This is a minimally invasive treatment that directs stem cells already present in tissues to heal damaged tissues.

LPL treatment was used to activate human dental stem cells in a laboratory culture system. Upon LPL treatment, the dental stem cells began to synthesize a powerful growth factor called transforming growth factor–β1 (TGF-β1). The endogenous synthesis of TGF-β1 and its receptor drove the dental stem cells to form dentin tubes.

When Kulkami and Mooney used an assay in animals called a “pulp capping model,” they discovered that LPL-activated dental stem cells were able to regenerate dentin after laser activation. To further demonstrate that these regenerative effects were the result of TGF-β1, Kalkami and Mooney and others made cells that did not have a functional TGF-β receptor II. This mutation completely abrogated the effects of LPL treatments. Also, if the dental stem cells were incubated with a TGF-βRI inhibitor, the effects of LPL on the dental stem cells was attenuated.

Thus, there is a simple and non-invasive way to activate a resident stem cell population in our bodies. Furthermore, the mechanisms by which LPL activates these stem cells has been defined as TGF-β mediated. These experiments also outlines the mechanism by which resident stem cells might be harnessed by means of light-activated endogenous cues for clinical regenerative applications. Exciting, huh?

Radio Interview About my New Book

I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

Gum-Based Stem Cells For Regenerative Medicine

The gums are also known as the gingivae, and this soft tissue serves as a biological barrier that covers the oral cavity of the maxillae and mandible (upper and lower jawbones). The gingivae also harbor a stem cell population known as gingival mesenchymal stem cells or GMSCs.

“Oh that’s a big surprise,” you say, “another mesenchymal stem cell population found in the body.” Well this one is a big deal because of its tissue of origin. Most MSCs are formed during embryonic development from cells that originate from the mesoderm, the embryonic tissue that lies between the skin of the embryo and the gut. Mesoderm forms the muscles, bones, connective tissue, adrenal glands, circulatory system, kidneys, gonads, and some other vitally important tissues.


However, in the head, a large number of tissues are formed from “neural crest cells.” Neural crest cells hail from the top of the neural tube, which is the beginnings of the spinal cord. The dorsal-most portion of the neural tube contains a population of cells that move out of the neural tube and colonize the embryo to form a whole host of tissues. These include: Neurons, including sensory ganglia, sympathetic and parasympathetic ganglia, and plexuses, Neuroglial cells, Schwann cells, Adrenal medulla, Calcitonin-secreting cells, Carotid body type I cells, Epidermal pigment cells, Facial cartilage and bone Facial and anterior ventral skull cartilage and bones, Corneal endothelium and stroma, Tooth papillae, Dermis, smooth muscle, and adipose tissue of skin of head and neck, Connective tissue of salivary, lachrymal, thymus, thyroid, and pituitary glands, Connective tissue and smooth muscle in arteries of aortic arch origin. Wow, that’s a lot of stuff. I think you can see that these neural crest cells are important players during embryonic development.


Songtao Shi, from the Ostrow School of Dentistry, University of Southern California and his co-workers demonstrated that approximately 90% of GMSCs are derived from cranial neural crest cells and 10% are derived from mesoderm. This is important because neural crest-based stem cells seem to have greater plasticity.

Shi and his team compared mesodermally derived MSCs with GMSCs and the neural crest derived MSCs have a greater ability to differentiate into neural cells and cartilage-making cells.

In a mouse model of colitis in which mice are fed dextran sulfate sodium, which induces colitis in the mice, the neural crest derived MSCs did a better job of relieving the inflammation associated with colitis than their mesodermally derived counterparts.

Shi admits that further research on these stem cells must be done in order to better understand them and their functional roles. Shi is especially interested in the functional interaction between the neural crest derived MSCs in the gum and the mesodermally derived MSCs. Also, their potential for suppressing inflammation in particular diseases of the immune system and wound healing needs to be examined in some detail.

Bmi1 Controls Adult Stem Cell “Stemness”

Stem cell scientists from the laboratory of Ophir Klein at UC San Francisco have discovered a new role for a protein called Bmi1 that might give clues as to how to get adult stem cells to regenerate organs.

Ophir Klein, the director of the Craniofacial and Mesenchymal Biology Program and chairman of the Division of Craniofacial Anomalies at UC San Francisco, said “Scientists have known that Bmi1 is a central control switch within the adult stem cells of many tissues, including the brain, blood, lung and mammary gland. Bmi1 also is a cancer-causing gene that becomes reactivated in cancer cells.”

Crystal structure of the BMI1 protein
Crystal structure of the BMI1 protein

All stem cells are somewhat immature in comparison to their adult counterparts. Stem cells also have the capacity to divide almost indefinitely and generate specialized cells. Bmi1 acts as a molecular switch that, if pushed in one direction, drives stem cells to proliferate and grow, but if pushed in the opposite direction, keeps cell proliferation in check. Research from Klein’s lab now suggests that Bmi1 might prevent the progeny of stem cells from differentiating into the wrong cell types in the wrong location.

Downstream targets of Bmi1
Downstream targets of Bmi1

This new discovery suggests that manipulation of Bmi1 and other regulatory molecules might be some of the steps included in laboratory recipes to turn specialized cell development on and off to create new cell-based treatments for tissue lost to injury, disease, or aging.

Also, the dual role of Bmi1 in pathological settings might be intriguing. Cancers are, in many cases, driven by adult stem cells that behave abnormally. If these stem cells could be differentiated, then their growth would slow. Possibly, inactivating Bmi1 in tumor stem cells might be one strategy.

In these experiments, Klein and his colleagues examined those adult stem cells found in the large incisors of mice. Unlike humans, these teeth grow continuously and are, therefore, an attractive model for stem cell research. Klein explained, “There is a large population of stem cells, and the way the daughter cells of the stem cells are produced is easy to track – it’s if they are on a conveyor belt.” Early in life, human beings possess a stem cell population that similarly drive tooth development, but they become inactive after the adult teeth are fully formed during early childhood.

Mouse mandible showing  the large, paired incisors
Mouse mandible showing the large, paired incisors

In the current study, postdoctoral research fellows Brian Biehs and Jimmy Hu showed that at the base of the growing mouse incisor there is a stem cell population that actively expresses Bmi1. In these cells, Bmi1 suppressed a set of genes called Hox genes. When activated, the Hox genes trigger the development of specific cell types and body structures.

In the mouse incisor, Bmi1 keeps these stem cells in their stem cell state and prevent them from differentiating prematurely or inappropriately. “This new knowledge is useful in a fundamental way for understanding how cell differentiating is controlled and may help us manipulate stem cells to get them to do what we want to do,” said Klein.

As they state in the abstract of their paper: “As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.”

Thus this finding from the Klein lab may provide a vital clue for the manipulation of adult stem cells and, perhaps, cancer cells.

Regeneration of Tooth Roots With Borrowed Stem Cells in Pigs

Because a recent post about tooth-making stem cells in alligators generated so much interest, I found another recent paper that reports the regeneration of the tooth root structure in pigs. This is a proof-of-concept paper that demonstrated the feasibility of such a procedure.

The journal is Stem Cells and Development and the research team is from the Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction in Beijing, China. The corresponding author is Songlin Wang from the Molecular Laboratory for Gene Therapy and Tooth Regeneration.

Tooth loss represents a growing problem in an aging population. Dental implants provide one solution, but without a good jaw bone into which these implants can be attached, implants have little chance of staying put. Regenerating a tooth root that can support a natural or artificial crown is the most important part of the tooth in maintaining tooth function.

In previous work, Wang and his collaborator Songtao Shi from UCLA have shown that stem cells from root apical papilla and periodontal ligament stem cells from exfoliated teeth can coat bioengineered surfaces and form tooth structures that can support artificial crowns in miniature pigs (see Sonoyama et al., PLoS One 1:e79-e92). However, aged patients sometimes have bone marrow stem cells that do not grow well in culture and respond poorly to bioengineering protocols. Therefore, Wang and his crew sought to demonstrate that mesenchymal stem cells from donor animals (allogeneic stem cells) could provide the same kind of benefit.

The two stem cell populations used in this paper was dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs). The DPSCs were cultured from exfoliated minipig teeth and grown in culture for two or three passages. The culture medium used, as far as I can tell, was the same one used the Gronthos in his PNAS paper that reported the isolation and characterization of DPSCs. That medium was a modified Eagle’s medium supplemented with 20% Fetal Calf Serum and 100 μM L-ascorbic acid 2-phosphate, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Gronthos then grew his cells at 37°C in 5% CO2 (see S. Gronthos, et al PNAS 97(25): 13625–13630). After 2-3 passages, the DPSCs were seeded on a hydroxyapatite tricalcium phosphate scaffold and grown in a bioreactor for 5-7 days

PDLSCs were grown in culture with approximately the same cocktail as the DPSCs and then plated on 60 mm dishes with vinylene carbonate (Vc). Vc induces the PDLSCs to grow s sheets that could be used to wrap the hydroxyapatite tricalcium phosphate structures that had been seeded with DPSCs.

These wrapped structures were implanted into the gums of minipigs and then used to secure dental implants.

Tooth Root Regeneration

After 6 months, the implants were assessed as was the integrity and strength of the surrounding tissue.

Gross, radiographic, and histological analysis of the bio-root 6 months after transplantation. (A, C) Gross view of the general shape of HA/TCP and the bio-root 6 months after transplantation (ellipse). (B, D) X-rays revealed that HA/TCP formed tissues without an obvious dental structure (ellipses), but the HA/TCP/DPSC/PDLSC sheet implant formed a hard root structure (ellipses). (E, F) No obvious boundary was observed between newly regenerated tissue and bone in the microcomputed tomography scan image of the HA/TCP group. (G, H) A hard root structure (arrows) was present and a clear PDL space found between the implant and surrounding bony tissue (triangle arrows). (I–K) HE staining showed some bone formation and HA/TCP remaining in the HA/TCP group (I), and PDL-like tissues were generated parallel to the dentin-like matrix structure in the autologous group (J) and allogeneic group (K). (L) Semiquantitative analysis showed that mineralized tissue regeneration capacity of autologous or allogeneic groups was significantly higher compared with the HA/TCP group. Percentage of mineralized tissues at 6 months after crown restoration was significantly higher than that before crown restoration in both autologous and allogeneic groups. No significant difference of regenerated mineralized tissue percentages was found between autologous and allogeneic groups. Scale bar: (I–K) 200 μm. B, bone; HA/TCP, hydroxyapatite/tricalcium phosphate; PDL, periodontal ligament; MT, mineralized tissue. *P<0.01 compared with autologous or allogeneic groups; #P<0.01 compared with autologous or allogeneic groups after crown restoration.
Gross, radiographic, and histological analysis of the bio-root 6 months after transplantation. (A, C) Gross view of the general shape of HA/TCP and the bio-root 6 months after transplantation (ellipse). (B, D) X-rays revealed that HA/TCP formed tissues without an obvious dental structure (ellipses), but the HA/TCP/DPSC/PDLSC sheet implant formed a hard root structure (ellipses). (E, F) No obvious boundary was observed between newly regenerated tissue and bone in the microcomputed tomography scan image of the HA/TCP group. (G, H) A hard root structure (arrows) was present and a clear PDL space found between the implant and surrounding bony tissue (triangle arrows). (I–K) HE staining showed some bone formation and HA/TCP remaining in the HA/TCP group (I), and PDL-like tissues were generated parallel to the dentin-like matrix structure in the autologous group (J) and allogeneic group (K). (L) Semiquantitative analysis showed that mineralized tissue regeneration capacity of autologous or allogeneic groups was significantly higher compared with the HA/TCP group. Percentage of mineralized tissues at 6 months after crown restoration was significantly higher than that before crown restoration in both autologous and allogeneic groups. No significant difference of regenerated mineralized tissue percentages was found between autologous and allogeneic groups. Scale bar: (I–K) 200 μm. B, bone; HA/TCP, hydroxyapatite/tricalcium phosphate; PDL, periodontal ligament; MT, mineralized tissue. *P

As you can see in panel E and F, control implants that had no cells and only hydroxyapatite calcium triphosphate showed no tooth-like structures, but in G and F, the structures with cells showed a consistent tooth structure with a periodontal ligament (see broad arrow).  In panels J and K, there is obvious bone formation with periodontal ligament in the autologous and allogeneic stem cell transplants.

Cross sections of the implants also showed that not only did these structures look like real tooth root structures, but they contained structures proteins indicative of real tooth root structures.  Dentin sialophosphoprotein (mercifully abbreviated to DSPP) is present in the cell-seeded implants, but in on the hydroxyapatite calcium triphosphate-only implants.

Clinical assessment of implants failed to detect any gingivitis or periodontal disease associated with the implants.

This experiment shows that stem cell-seeded scaffolds can regenerate tooth root structures.  Since this worked in minipigs and not simply rodents, these results strongly suggest that such a strategy could work in humans.  Clinical trials anyone?

Alligator Stem Cells and Tooth Replacement

Mammals usually have one set of baby teeth (also known as milk teeth) and after those are lost, we have one set of adult teeth and these are not replaced if they are lost. This condition is called “monophyodont.” Reptiles and sharks, however constantly replace their teeth. This condition is called “polyphyodont.” Alligators and crocodiles are among one group of reptiles that replace their teeth throughout their lives, and because the development of these creatures has been studied to some extent, it is known that the ability of these creatures to replace their teeth on a regular basis results from a resident stem cell population. Studying that stem cell population more closely might provide clues for tooth replacement in humans.

American Alligator
American Alligator

A research team led by scientists at the Keck School of Medicine professor of pathology Cheng-Ming Chuong at the University of Southern California. Dr. Chuong and his collaborators from around the world have identified unique cellular and molecular mechanisms behind tooth renewals in American alligators.

Chuong explained, “Humans naturally have only two sets of teeth – baby teeth and adult teeth. Ultimately, we want to identify stem cells that can be used as a resource to stimulate tooth renewal in adult humans who have lost teeth. But, to do that, we must first understand how they renew in other animals and why they stop in people.”

Even though humans cannot replace their adult teeth, a tissue called the dental lamina remains, which is known to be crucial for tooth development.

Why are alligators potentially a good model system for tooth replacement in mammals? First author of this study, Ping Wu, explained it this way, “Alligator teeth are implanted in sockets of the dental bone, like human teeth. They have 80 teeth, each of which can be replaced up to 50 times over their lifetime, making them the ideal model for comparison to human teeth.”

Through the use of microscopic imaging techniques, Chuong and others found that each alligator tooth is a complex unit of three components: a functional tooth, a replacement tooth, and the dental lamina, all other which are at different developmental stages.

The tooth units are built to enable a smooth transition from dislodgement of the functional, mature tooth to replacement with a new tooth. Further imaging studies strongly suggested that the dental lamina contains a stem cell population from which new replacement teeth develop.

“Stem cells divide more slowly than other cells, said co-author Randall B. Widelitz, who serves as an associate professor of pathology at USC. Widelitz continued, “The cells in the alligator’s dental lamina behaved like we would expect stem cells to behave. In the future, we hope to isolate those cells from the dental lamina to see whether we can use them to regenerate teeth in the lab.”

The researchers also intend to learn what molecular networks are involved in repetitive renewal and hope to apply the principles to regenerative medicine in the future.

The authors also noted that novel cellular mechanisms are used during the development of the tooth unit. Also, unique molecular signaling speeds growth of replacement teeth when functional teeth are lost.

See P. Wu PNAS 2013; DOI: 10.1073/pnas.12132110.

Stem Cells From Gum Tissue Help Replace Missing Teeth

Researchers from King’s College London, UK have developed a new method that replaced missing teeth with bioengineered material made from a patient’s own gum cells.

If a patient loses a tooth, the dentist or oral surgeon will typically replace it with an implant. The vast majority of dental implants used today are root-form endosseous implants. Such implants have a similar look to an actual tooth root and are placed within the bone of the jaw. The bone of the jaw fuses the surface of the implant with the surrounding bone (a process known as osseointegration). Because dental implants lack the periodontal ligament they will feel slightly different from natural teeth during chewing. Also, the friction from chewing and from other jaw movements can cause loss of bone around the implant.


Research by members of Paul Sharpe‘s laboratory at King’s College London has brought us closer to the reality of bioengineered teeth to replace toss teeth. Bioengineered tooth research has focussed primarily on producing immature teeth that can grow into adult teeth. Typically, such tooth buds are grown in culture and then transplanted into the gums. The gum actually provides and adequate environment for embryonic tooth buds to develop and form adult teeth. Therefore, the prospect of forming bioteeth certainly seems viable. The only question is identifying the cells and materials that can combine to properly form a normal adult tooth.


Sharpe noted, “What is required is the identification of adult sources of human epithelial and mesenchymal cells that can be obtained in sufficient numbers to make biotooth formation a viable alternative to dental implants.”

Sharpe and his colleagues surmised that gum tissue might provide the right cells for this project. Therefore, they isolated adult human gum tissue samples from patients at the Dental Institute at King’s College and grew it in culture in the laboratory. Next, Sharpe’s group combined this gum tissue with mouse embryonic tooth mesenchyme cells, which are stem cells that can induce tooth formation.  This gum-tooth combination created teeth with surrounding gum tissue that could be transplanted into the mouths of mice. The teeth had dentine, enamel and viable roots.

The epithelial cells from human gum were able to respond to tooth-inducing signals from the embryonic tooth mesenchymal cells in a manner that allowed them to contribute to the tooth crown and the roots, and formed all the available cell types necessary for normal tooth formation. Thus, it appears that gum biopsies can provide a realistic source for human biotooth production.

The next step in this research is the formidable challenge of finding a mesenchymal stem cell population that can induce tooth formation. Presently, only embryonic mesenchymal cells can do this, according to Sharpe, but it is possible that adult mesenchymal stem cells can be manipulated to become tooth-inducing cells.

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

First Human Study Using Dental Stem Cells

This is an old paper, but it is still a good read.

November 12th 2009 the first clinical study involving human dental stem cells was published in the journal European Cells and Materials journal.

This study examined patients with impacted wisdom teeth who also had bone loss (resorption) at the site of impaction. Such a bone defect does not repair on its own after the wisdom teeth are removed. Therefore, the researchers used a mixture of dental pulp stem cells harvested from the patient’s non-impacted, upper wisdom teeth and placed them onto a “scaffold” made of collagen sponge. They then used this mixture to fill in the injured areas that remained after the impacted teeth were removed from the lower jaw. The area in the upper jaw served as a control, or comparison, since no dental stem cells were used in that region.

Three months after treatment, the bone had completely regenerated at the injury site and the periodontal tissue had been restored. In the seven patients who returned for one-year follow-up examinations, optimal bone regeneration was observed. The investigators concluded that dental stem cells embedded onto a collagen sponge scaffold can completely restore bone defects in the human jaw. Furthermore, these cells have the potential to repair and/or regenerate tissues and organs.

Before the publication of this paper, jaw defects had been repaired using dental stem cells in an animal model, but never in humans. In fact, no dental stem cell therapies have ever been used in human patients. This bone grafting study is very exciting for the future promise of dental stem cell therapies. It does not matter if the dental stem cells come from a dental stem cell bank, such as the National Dental Pulp Laboratory, or individuals who wish to preserve their own or their children’s pulp in order to have a source of stem cells that they might be able to put to use for future medical needs.

Sox2 Marks Incisor Stem Cells

Finnish stem cell researchers have discovered a gene that serves as a marker for front teeth. Researchers in the group of Professor Irma Thesleff at the Institute of Biotechnology in Helsinki, Finland have developed a method to record the division, movement, and specification of these dental stem cells. Apparently, building a tooth requires a detailed recipe to instruct cells to differentiate towards proper lineages and form dental cells.

Building a tooth from stem cells is a very difficult talk. However the development of new bioengineering protocols might make this possible in a few years. There is definitely a demand for tissue engineered teeth, since tooth loss affects oral health, quality of life, and your appearance. To build a tooth, a detailed recipe to instruct cells to direct cells to differentiate towards proper cell lineages and form dental cells is needed. However, in order to study of stem cells, scientists need a specific protein that only those cells make (a marker), that allows the isolation of and purification of dental stem cells. Unfortunately, the lack of an identifiable marker has hindered such studies so far.

The mouse system is an excellent system for such studies, since mouse incisors grow continuously throughout life and this growth is fueled by stem cells located at the base of the tooth. In Professor Thesleff’s lab, her students traced the descendants of genetically labeled cells, and showed that a gene called Sox2 labels stem cells that give rise to enamel-forming ameloblasts as well as other cell lineages of the tooth.

Even though human teeth don’t grow continuously, the mechanisms that control and regulate their growth are similar to those in mouse teeth. Therefore, the discovery of Sox2 as a marker for dental stem cells is an important step toward developing a complete bioengineered tooth.

In the future, it may be possible to grow new teeth from stem cells to replace lost ones, said researcher Emma Juuri, a co-author of the study.