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Thread: Tooth pulp stem cells regenerate spinal cord

  1. #11

    Response to J Silver

    Sakai, et al. [1] studied the effects of transplanted dental pulp derived stem cells (DPSC from adult human teeth) and exfoliated deciduous teeth (SHEDS from young human teeth) on transected rat spinal cords. Before discussing this paper, let me first give some background.

    Teeth have stem cells. In 1999, Harada, et al. [2] from Finland analyzed cells in mouse incisors and found cells that take up 5-BrdU and DiI. The cells reside in the cervical loop epithelium, consisting of a central core of stellate reticulum cells surrounded by a layer of basal epithelial cells. These cells produce ameloblasts that form enamel. Some of the stellate cells express Notch1 (a stem cell factor) and the mesenchyme underlying the basal epithelia produce FGF growth factors, which modulate Notch in the cells. In subsequent studies, Harada, et al. [3] showed that the stem cells divide slowly giving rise to two daughter cells, one of which differentiates into ameloblasts and FGF10 plays a role in maintaining this stem cell compartment [4].

    In 2001, Nosrat, et al. [5] reported the dental pulp cells in growing mouse incisors produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury. The following year, Nakashima, et al. [6] reported that the growth and differentiation factor GDF11 induced differentiation of the dental pulp stem cells into odontoblasts. In 2003, Miura, et al. [7] published a paper in the Proceedings of the National Academy of Science naming stem cells obtained from dental pulp can differentiate into a variety of cells, including neural cells, adipocytes, and odontoblasts. These findings were subsequently publicized in popular articles with headlines such as “Supply of stem cells found in baby teeth” [8] and “Primary teeth found to be a source of stem cells”.

    Seo, et al. [9] suggested that these stem cells are mesenchymal stem cells that can differentiate into a variety of cells. These cells can be isolated, cultured, expanded from healthy subjects. About 30% of the cells in extracted premolar or wisdom teeth are multipotent [10]. Sonoyama, et al. [11] identified mesenchymal stem cells from the root apical papilla and called them stem cell isolated from apical papilla (SCAP). Zhang, et al. [12] showed that these cells can be cryopreserved. Fang, et al. [13] used these cells for plastic surgery. Jo, et al. [14] described methods to isolate and culture CD29+ and CD44 + mesenchymal stem cells from adult human tooth pulp. Wang, et al. [15] described factors that regulate these cells. Finally, in 2008, Arthur, et al., [16] reported that adult human dental pulp stem cells could produce functionally active neurons.

    Several investigators have used dental pulp stem cells to treat a variety of conditions. In 2008, Kerkis, et al. [17] used these cells to treat golden retrievers with muscular dystrophy with some positive results. Other investigators have reported positive results of such cells transplanted into injured rat spinal cords in 2011. For example, Taghipour, et al. [18] recently transplanted DPDS cells into animals with contused spinal cords and found that the cells survived and differentiated into neurons, astrocytes, and oligodendroglia, and improved locomotor recovery. De Almeida, et al., [19] transplanted DPDS cells into young adult female C57/BL6 mice that were injured with a vascular clip for 1 minute. The cells were transplanted at 7 or 28 days and the authors observed better white matter preservation than vehicle controls, higher trophic factor expression in the tissues, better tissue organization, and the presence of many axons myelinated by Schwann cells or oligodendrocytes.

    Given this background, does this paper from Sakai, et al. [1] report novel and interesting findings? I believe so.

    First, Sakai, et al. compared the results of SHEDS (these are young teeth, 6-12 years old) versus DPDS (these are adult teeth pulp, 18-30 years old) and provided detailed flow cytometry markers of the cells. Over 90% of these cells are CD90, CD73, and CD105 positive. They are not CD45, CD34, CD11b, or HLA-DR positive; these are usually markers that are associated with hematopoietic or mononuclear cells. The fact that they don’t express HLA-DR is of interest because this is one of the important tissue compatibility antigens that lead to immune rejection if not matched. Finally, these DPDS cells express nestin (a neural stem cell marker) and other markers of differentiated neural cells including GFAP (for differentiated astrocytes), beta-III tubulin and NeuN (for differentiated neurons), A2B5 (a glial-restricted precursor marker), and MBP (or myelin basic protein which is associated with myelin). So, these cells from teeth express markers of neural stem cells.

    Second, the authors did experiments suggesting SHEDS cells regenerated corticospinal tract and raphe spinal serotonergic axons. To show the former, they injected a tracer called biotinylated dextran amine (BDA) into the cortex, waited for the cortical neurons to transport the tracer into axons going into the spinal cord, and then looked for the tracer in axons below the injury site. One must remember that they transected the spinal cords, one of the most unfavorable injuries that I can imagine for the spinal cord. I am surprised that any BDA is present in the spinal cord. Finally, they showed that there were serotonergic axons in the spinal cord below the injury site. Normally, all serotonergic axons come from the brainstem and there should be no serotonergic fibers below the injury site. Admittedly, their histology is not the best of quality (they only show one figure of the corticospinal tract regeneration) that suggested presence of BDA tracer in the spinal cord below the injury site (figure 3). They did show that there were more neurofilament labelled axons on both the rostral and caudal stump (figure 2). There were more myelinated axons in SHEDS treated animals (figure 6). Finally, there were more serotonergic fibers in the proximal and distal spinal cords of the transplanted animals (figure 4). None of these findings by themselves are convincing but together I think that there are more axons in transplanted spinal cords and some of the axons are crossing the injury site. I don’t share Jerry Silver’s strong opinion that this paper should not have been published.

    Third, the BBB score recovery by the transplanted animals was convincing to me. Normally, when you transected the spinal cord of rats, you end up with BBB scores of <2 after 6-8 weeks. In their control animals, they got BBB scores of less than one. Their DPDS treated animals had BBB scores of 3. However, the mean BBB scores of the rats treated with SHEDS (the stem cells from younger teeth) nearly reached 8. The standard errors of means are small (less than one BBB score). It is true that a BBB score of 8 or less does not mean that the animals were doing weight-supported walking. On the other hand, it means that the animals were engaged in sweeping and 3 joint movements of their legs. This is actually rare in rats with complete spinal cord injury transections. I have seen many hundreds of rats with BBB scores and I have never seen transected rats do this.

    In my opinion, the authors have taken one of the most severe spinal cord injury models and showed that transplantation of two types of cells result in evidence of regeneration and recovery of hindlimb movements. The histological and behavioral evidence support each other. The type of cells that produce less histological improvement stimulated less recovery. The authors showed that the SHED cells seem to support axonal growth in culture better and this is interesting as well. To me, this paper deserves publication. It is all right if some people decide that they are not convinced but the paper needs to see the light of day instead of being buried. This debate is good. To tell you the truth, I don’t think that the data for chondroitinase efficacy for spinal cord injury is that much better.

    Two other papers, i.e. Taghipour, et al. [18] and De Almeida, et al., [19] have reported beneficial effects, although neither paper are as good as this one by Sakai, et al. [1]. I happen to know of one other laboratory that has shown beneficial effects of SHEDS cells in a rat contusion model and hopefully will publish the results soon. It is particularly interesting that SHEDS cells, from younger teeth, appear to be significantly better than DPDS from older teeth. Are these cells worthwhile considering for clinical trial in chronic spinal cord injury? I want to emphasize that this was an acute spinal cord injury therapy. The cells were injected into the rostral and caudal stumps shortly after transection. There should be a study of SHEDS transplants into chronic spinal cord contusion injury. Also, there is the question of whether one can get SHEDS from HLA-matched young teeth for transplantation. But, despite these caveats, I think this is a very interesting therapy that we need to keep in mind as we consider candidates for trial.

    References Cited
    1. Sakai K, Yamamoto A, Matsubara K, Nakamura S, Naruse M, Yamagata M, Sakamoto K, Tauchi R, Wakao N, Imagama S, Hibi H, Kadomatsu K, Ishiguro N and Ueda M (2011). Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms. The Journal of clinical investigation Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.
    2. Harada H, Kettunen P, Jung HS, Mustonen T, Wang YA and Thesleff I (1999). Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. The Journal of cell biology 147: 105-20. Developmental Biology Programme, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, 00014 Helsinki, Finland. The continuously growing mouse incisor is an excellent model to analyze the mechanisms for stem cell lineage. We designed an organ culture method for the apical end of the incisor and analyzed the epithelial cell lineage by 5-bromo-2'-deoxyuridine and DiI labeling. Our results indicate that stem cells reside in the cervical loop epithelium consisting of a central core of stellate reticulum cells surrounded by a layer of basal epithelial cells, and that they give rise to transit-amplifying progeny differentiating into enamel forming ameloblasts. We identified slowly dividing cells among the Notch1-expressing stellate reticulum cells in specific locations near the basal epithelial cells expressing lunatic fringe, a secretory molecule modulating Notch signaling. It is known from tissue recombination studies that in the mouse incisor the mesenchyme regulates the continuous growth of epithelium. Expression of Fgf-3 and Fgf-10 were restricted to the mesenchyme underlying the basal epithelial cells and the transit-amplifying cells expressing their receptors Fgfr1b and Fgfr2b. When FGF-10 protein was applied with beads on the cultured cervical loop epithelium it stimulated cell proliferation as well as expression of lunatic fringe. We present a model in which FGF signaling from the mesenchyme regulates the Notch pathway in dental epithelial stem cells via stimulation of lunatic fringe expression and, thereby, has a central role in coupling the mitogenesis and fate decision of stem cells.
    3. Harada H, Mitsuyasu T, Toyono T and Toyoshima K (2002). Epithelial stem cells in teeth. Odontology / the Society of the Nippon Dental University 90: 1-6. Second Department of Oral Anatomy and Cell Biology, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan. Many tissues and organs maintain a process known as homeostasis, in which cells are replenished as they die as a result of apoptosis or injury. The continuously growing mouse incisors are an excellent model for studying the molecular mechanisms of cell homeostasis, renewal, and repair. We elucidated these mechanisms in mouse incisors by detecting adult stem cells and analyzing the stem cell lineage by bromodeoxyuridine (BrdU) labeling analysis. The stem cells divide slowly, giving rise to a daughter cell that remains in the cervical loop and a second daughter cell that enters the zone of rapidly dividing inner enamel epithelial cells (transit-amplifying cell population). During subsequent rounds of cell division, the latter cells move toward the incisal end and differentiate into ameloblasts that form the enamel matrix. Recent evidence from gene knockout mice suggests that fibroblast growth factor (Fgf10) plays an important role in the formation and maintenance of stem cells in the development of mouse incisors. The role of dental stem cells in odontogenic tumors is discussed.
    4. Harada H, Toyono T, Toyoshima K, Yamasaki M, Itoh N, Kato S, Sekine K and Ohuchi H (2002). FGF10 maintains stem cell compartment in developing mouse incisors. Development 129: 1533-41. Second Department of Oral Anatomy and Cell Biology, Kyushu Dental College, 2-6-1, Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan. Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Recently, a mouse incisor was used as a model to show that fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult stem cells. To further illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, we have analyzed incisor development in Fgf10-deficient mice and have examined the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. We found that the absence of the cervical loop was due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, we estimated the growth of dental epithelium from incisor explants of FGF10-null mice by organ culture. The dental epithelium of FGF10-null mice showed limited growth, although the epithelium of wild-type mice appeared to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induced apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescued the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs.
    5. Nosrat IV, Widenfalk J, Olson L and Nosrat CA (2001). Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury. Dev Biol 238: 120-32. Laboratory of Oral Neurobiology, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109, USA. Interactions between ingrowing nerve fibers and their target tissues form the basis for functional connectivity with the central nervous system. Studies of the developing dental pulp innervation by nerve fibers from the trigeminal ganglion is an excellent example of nerve-target tissue interactions and will allow specific questions regarding development of the dental pulp nerve system to be addressed. Dental pulp cells (DPC) produce an array of neurotrophic factors during development, suggesting that these proteins might be involved in supporting trigeminal nerve fibers that innervate the dental pulp. We have established an in vitro culture system to study the interactions between the dental pulp cells and trigeminal neurons. We show that dental pulp cells produce several neurotrophic factors in culture. When DPC are cocultured with trigeminal neurons, they promote survival and a specific and elaborate neurite outgrowth pattern from trigeminal neurons, whereas skin fibroblasts do not provide a similar support. In addition, we show that dental pulp tissue becomes innervated when transplanted ectopically into the anterior chamber of the eye in rats, and upregulates the catecholaminergic nerve fiber density of the irises. Interestingly, grafting the dental pulp tissue into hemisected spinal cord increases the number of surviving motoneurons, indicating a functional bioactivity of the dental pulp-derived neurotrophic factors in vivo by rescuing motoneurons. Based on these findings, we propose that dental pulp-derived neurotrophic factors play an important role in orchestrating the dental pulp innervation.
    6. Nakashima M, Mizunuma K, Murakami T and Akamine A (2002). Induction of dental pulp stem cell differentiation into odontoblasts by electroporation-mediated gene delivery of growth/differentiation factor 11 (Gdf11). Gene therapy 9: 814-8. Department of Clinical Oral Molecular Biology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. The long-term goal of dental treatment is to preserve teeth and prolong their function. In dental caries an efficient method is to cap the exposed dental pulp and conserve the pulp tissue with reparative dentin. We examined whether growth/differentiation factor 11 (GDF11), a morphogen could enhance the healing potential of pulp tissue to induce differentiation of pulp stem cells into odontoblasts by electroporation-mediated gene delivery. Recombinant human GDF11 induced the expression of dentin sialoprotein (Dsp), a differentiation marker for odontoblasts, in mouse dental papilla mesenchyme in organ culture. The Gdf11 cDNA plasmid which was transferred into mesenchymal cells derived from mouse dental papilla by electroporation, induced the expression of Dsp. The in vivo transfer of Gdf11 by electroporation stimulated the reparative dentin formation during pulpal wound healing in canine teeth. These results provide the scientific basis and rationale for gene therapy for endodontic treatments in oral medicine and dentistry.
    7. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG and Shi S (2003). SHED: stem cells from human exfoliated deciduous teeth. Proceedings of the National Academy of Sciences of the United States of America 100: 5807-12. Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. To isolate high-quality human postnatal stem cells from accessible resources is an important goal for stem-cell research. In this study we found that exfoliated human deciduous tooth contains multipotent stem cells [stem cells from human exfoliated deciduous teeth (SHED)]. SHED were identified to be a population of highly proliferative, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts. After in vivo transplantation, SHED were found to be able to induce bone formation, generate dentin, and survive in mouse brain along with expression of neural markers. Here we show that a naturally exfoliated human organ contains a population of stem cells that are completely different from previously identified stem cells. SHED are not only derived from a very accessible tissue resource but are also capable of providing enough cells for potential clinical application. Thus, exfoliated teeth may be an unexpected unique resource for stem-cell therapies including autologous stem-cell transplantation and tissue engineering.
    8. (2003). Supply of stem cells found in baby teeth. Dentistry today 22: 26, 28.
    9. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY and Shi S (2004). Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364: 149-55. Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA. BACKGROUND: Periodontal diseases that lead to the destruction of periodontal tissues--including periodontal ligament (PDL), cementum, and bone--are a major cause of tooth loss in adults and are a substantial public-health burden worldwide. PDL is a specialised connective tissue that connects cementum and alveolar bone to maintain and support teeth in situ and preserve tissue homoeostasis. We investigated the notion that human PDL contains stem cells that could be used to regenerate periodontal tissue. METHODS: PDL tissue was obtained from 25 surgically extracted human third molars and used to isolate PDL stem cells (PDLSCs) by single-colony selection and magnetic activated cell sorting. Immunohistochemical staining, RT-PCR, and northern and western blot analyses were used to identify putative stem-cell markers. Human PDLSCs were transplanted into immunocompromised mice (n=12) and rats (n=6) to assess capacity for tissue regeneration and periodontal repair. Findings PDLSCs expressed the mesenchymal stem-cell markers STRO-1 and CD146/MUC18. Under defined culture conditions, PDLSCs differentiated into cementoblast-like cells, adipocytes, and collagen-forming cells. When transplanted into immunocompromised rodents, PDLSCs showed the capacity to generate a cementum/PDL-like structure and contribute to periodontal tissue repair. INTERPRETATION: Our findings suggest that PDL contains stem cells that have the potential to generate cementum/PDL-like tissue in vivo. Transplantation of these cells, which can be obtained from an easily accessible tissue resource and expanded ex vivo, might hold promise as a therapeutic approach for reconstruction of tissues destroyed by periodontal diseases.
    10. Nagatomo K, Komaki M, Sekiya I, Sakaguchi Y, Noguchi K, Oda S, Muneta T and Ishikawa I (2006). Stem cell properties of human periodontal ligament cells. Journal of periodontal research 41: 303-10. Periodontology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan. BACKGROUND AND OBJECTIVE: Stem cells have been used for regenerative therapies in various fields. The proportion of cells that possess stem cell properties in human periodontal ligament (PDL) cells is not yet well understood. In this study, we quantitatively characterized human PDL cells to clarify their stem cell properties, including self-renewal, multipotency, and stem cell marker expression. MATERIAL AND METHODS: PDL cells were obtained from extracted premolar or wisdom teeth, following which a proliferation assay for self-renewal, a differentiation assay for multipotency, immunostaining for STRO-1, and fluorescence-activated cell sorter (FACS) analysis for stem cell markers (including CD105, CD166, and STRO-1) were performed. RESULTS: Approximately 30% of 400 PDL cells were found to possess replicative potential and formed single-cell colonies, and 30% of these colonies displayed positive staining for STRO-1, 20% differentiated into adipocytes and 30% differentiated into osteoblasts. FACS analysis revealed that PDL cells, including cell populations, expressed the stem cell markers CD105, CD166, and STRO-1. CONCLUSION: The findings of this study indicated that PDL cells possess crucial stem cell properties, such as self-renewal and multipotency, and express the mesenchymal stem cell markers CD105, CD166, and STRO-1 on their cell surface, although there were some variations. Thus, PDL cells can be used for periodontal regenerative procedures.
    11. Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, Gronthos S, Wang CY, Wang S and Shi S (2006). Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS ONE 1: e79. Center for Craniofacial Molecular Biology, University of Southern California School of Dentistry, Los Angeles, California, United States of America; Department of Oral and Maxillofacial Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.
    12. Zhang W, Walboomers XF, Shi S, Fan M and Jansen JA (2006). Multilineage differentiation potential of stem cells derived from human dental pulp after cryopreservation. Tissue engineering 12: 2813-23. Radboud University Nijmegen Medical Centre, Periodontology & Biomaterials, Nijmegen, The Netherlands. The current study aimed to prove that human dental pulp stem cells (hDPSCs) isolated from the pulp of third molars can show multilineage differentiation after cryopreservation. First, hDPSC were isolated via enzymatic procedures, and frozen in liquid nitrogen until use. After defrosting, cells were analyzed for proliferative potential and the expression of the stem cell marker STRO-1. Subsequently, cells were cultured in neurogenic, osteogenic/odontogenic, adipogenic, myogenic, and chondrogenic inductive media, and analyzed on basis of morphology, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR) for specific marker genes. All data were replicated, and the results of the primary cells were compared to similar tests with an additional primary dental pulp stem cell strain, obtained from the National Institutes of Health (NIH). Results showed that our cell population could be maintained for at least 25 passages. The existence of stem/ progenitor cells in both cell strains was proven by the STRO-1 staining. Under the influence of the 5 different media, both cell strains were capable to advance into all 5 differentiation pathways. Still differences between both strains were found. In general, our primary culture performed better in myogenic differentiation, while the externally obtained cells were superior in the odontogenic/osteogenic and chondrogenic differentiation pathways. In conclusion, the pulp tissue of the third molar may serve as a suitable source of multipotent stem cells for future tissue engineering strategies and cell-based therapies, even after cryopreservation.
    13. Fang D, Seo BM, Liu Y, Sonoyama W, Yamaza T, Zhang C, Wang S and Shi S (2007). Transplantation of mesenchymal stem cells is an optimal approach for plastic surgery. Stem Cells 25: 1021-8. Salivary Gland Disease Center and the Molecular Laboratory for Gene Therapy, Capital Medical University School of Stomatology, Beijing, China. Mesenchymal stem cells (MSCs) are able to differentiate into a variety of cell types, offering promising approaches for stem cell-mediated tissue regeneration. Here, we explored the potential of utilizing MSCs to reconstruct orofacial tissue, thereby altering the orofacial appearance. We demonstrated that bone marrow MSCs were capable of generating bone structures and bone-associated marrow elements on the surfaces of the orofacial bone. This resulted in significant recontouring of the facial appearance in mouse and swine. Notably, the newly formed bone and associated marrow tissues integrated with the surfaces of the recipient bones and re-established a functional bone marrow organ-like system. These data suggested that MSC-mediated tissue regeneration led to a body structure extension, with the re-establishment of all functional components necessary for maintaining the bone and associated marrow organ. In addition, we found that the subcutaneous transplantation of another population of MSCs, the human periodontal ligament stem cells (PDLSCs), could form substantial amounts of collagen fibers and improve facial wrinkles in mouse. By contrast, bone marrow MSCs failed to survive at 8 weeks post-transplantation under the conditions used for the PDLSC transplantation. This study suggested that the mutual interactions between donor MSCs and recipient microenvironment determine long-term outcome of the functional tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.
    14. Jo YY, Lee HJ, Kook SY, Choung HW, Park JY, Chung JH, Choung YH, Kim ES, Yang HC and Choung PH (2007). Isolation and characterization of postnatal stem cells from human dental tissues. Tissue engineering 13: 767-73. Department of Oral and Maxillofacial Surgery, Tooth Bioengineering National Research Lab, BK21, and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea. It was reported that postnatal stem cells are present in adult tissues such as bone marrow, liver, muscle, dental pulp, and periodontal ligament. We isolated postnatal stem cells from human dental tissues such as dental pulp (DPSC), periodontal ligament (PDLSC), periapical follicle (PAFSC), and the surrounding mandibular bone marrow (MBMSC) to ascertain their properties. Immunocytochemistry proved the existence of stem cells in these cell populations using STRO-1 as a stem cell marker. These cells also expressed the mesenchymal stem cell (MSC) markers CD29 and CD44. The isolated cells showed self-renewal capabilities and colony-forming efficiency. Almost all of the dental stem cells showed optimal growth when they were cultured in alpha modification of Eagle's medium (alpha-MEM) supplemented with 10% fetal calf serum (FCS) and 100 microM ascorbic acid. Only the PAFSC showed increased proliferation in 20% FCS and 50 microM ascorbic acid. All of the dental stem cells were capable of differentiating into adipocytes and mineral nodule forming cells. MBMSC, in particular, showed much better mineralization compared to the others. These results indicate that MSCs exist in various tissues of the teeth and can differentiate into osteoblasts, adipocytes, and other kinds of cells with varying efficiency.
    15. Wang XP, Suomalainen M, Felszeghy S, Zelarayan LC, Alonso MT, Plikus MV, Maas RL, Chuong CM, Schimmang T and Thesleff I (2007). An integrated gene regulatory network controls stem cell proliferation in teeth. PLoS biology 5: e159. Developmental Biology Programme, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland. Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.
    16. Arthur A, Rychkov G, Shi S, Koblar SA and Gronthos S (2008). Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells 26: 1787-95. The Australian Research Council, Centre for the Molecular Genetics of Development, University of Adelaide, Adelaide, Australia. Human adult dental pulp stem cells (DPSCs) reside within the perivascular niche of dental pulp and are thought to originate from migrating cranial neural crest (CNC) cells. During embryonic development, CNC cells differentiate into a wide variety of cell types, including neurons of the peripheral nervous system. Previously, we have demonstrated that DPSCs derived from adult human third molar teeth differentiate into cell types reminiscent of CNC embryonic ontology. We hypothesized that DPSCs exposed to the appropriate environmental cues would differentiate into functionally active neurons. The data demonstrated that ex vivo-expanded human adult DPSCs responded to neuronal inductive conditions both in vitro and in vivo. Human adult DPSCs, but not human foreskin fibroblasts (HFFs), acquired a neuronal morphology, and expressed neuronal-specific markers at both the gene and protein levels. Culture-expanded DPSCs also exhibited the capacity to produce a sodium current consistent with functional neuronal cells when exposed to neuronal inductive media. Furthermore, the response of human DPSCs and HFFs to endogenous neuronal environmental cues was determined in vivo using an avian xenotransplantation assay. DPSCs expressed neuronal markers and acquired a neuronal morphology following transplantation into the mesencephalon of embryonic day-2 chicken embryo, whereas HFFs maintained a thin spindle fibroblastic morphology. We propose that adult human DPSCs provide a readily accessible source of exogenous stem/precursor cells that have the potential for use in cell-therapeutic paradigms to treat neurological disease.
    17. Kerkis I, Ambrosio CE, Kerkis A, Martins DS, Zucconi E, Fonseca SA, Cabral RM, Maranduba CM, Gaiad TP, Morini AC, Vieira NM, Brolio MP, Sant'Anna OA, Miglino MA and Zatz M (2008). Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic? Journal of translational medicine 6: 35. Laboratorio de Genetica e Imunoquimica, Instituto Butantan, Sao Paulo, Brasil. BACKGROUND: The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. METHODS: Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. RESULTS AND DISCUSSION: We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. CONCLUSION: Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.
    18. Taghipour Z, Karbalaie K, Kiani A, Niapour A, Bahramian H, Nasr-Esfahani MH and Baharvand H (2011). Transplantation of Undifferentiated and Induced Human Exfoliated Deciduous Teeth-Derived Stem Cells Promote Functional Recovery of Rat Spinal Cord Contusion Injury Model. Stem cells and development 1 Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology , ACECR, Isfahan, Iran . Regarding both the neural crest origin and neuronal potential of stem cells from human exfoliated deciduous teeth (SHED), here, we assessed their potential in addition to neural induced SHED (iSHED) for functional recovery when transplanted in a rat model for acute contused spinal cord injury (SCI). Following transplantation, a significant functional recovery was observed in both groups relative to the vehicle and control groups as determined by the open field locomotor functional test. We also observed that animals that received iSHED were in a better state as compared with the SHED group. Immunohistofluorescence evaluation 5 weeks after transplantation showed neuronal and glial differentiation and limited proliferation in both groups. However, myelin basic protein and chondroitin sulfate proteoglycan NG2-oligodendrocyte markers-were increased and glial fibrillary acidic protein-astrocyte marker-was decreased in the iSHED group in comparison with the SHED group. These findings have demonstrated that transplantation of SHED or its derivatives could be a suitable candidate for the treatment of SCI as well as other neuronal degenerative diseases.
    19. de Almeida FM, Marques SA, Ramalho Bdos S, Rodrigues RF, Cadilhe DV, Furtado D, Kerkis I, Pereira LV, Rehen SK and Martinez AM (2011). Human dental pulp cells: a new source of cell therapy in a mouse model of compressive spinal cord injury. Journal of Neurotrauma 28: 1939-49. Programa de Pesquisa em Neurociencia Basica e Clinica, Instituto de Ciencias Biomedicas, Centro de Ciencias da Saude, Universidade Federal do Rio de Janeiro, Brazil. Strategies aimed at improving spinal cord regeneration after trauma are still challenging neurologists and neuroscientists throughout the world. Many cell-based therapies have been tested, with limited success in terms of functional outcome. In this study, we investigated the effects of human dental pulp cells (HDPCs) in a mouse model of compressive spinal cord injury (SCI). These cells present some advantages, such as the ease of the extraction process, and expression of trophic factors and embryonic markers from both ecto-mesenchymal and mesenchymal components. Young adult female C57/BL6 mice were subjected to laminectomy at T9 and compression of the spinal cord with a vascular clip for 1 min. The cells were transplanted 7 days or 28 days after the lesion, in order to compare the recovery when treatment is applied in a subacute or chronic phase. We performed quantitative analyses of white-matter preservation, trophic-factor expression and quantification, and ultrastructural and functional analysis. Our results for the HDPC-transplanted animals showed better white-matter preservation than the DMEM groups, higher levels of trophic-factor expression in the tissue, better tissue organization, and the presence of many axons being myelinated by either Schwann cells or oligodendrocytes, in addition to the presence of some healthy-appearing intact neurons with synapse contacts on their cell bodies. We also demonstrated that HDPCs were able to express some glial markers such as GFAP and S-100. The functional analysis also showed locomotor improvement in these animals. Based on these findings, we propose that HDPCs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.
    Last edited by Wise Young; 12-08-2011 at 08:05 PM.

  2. #12
    One thing that amazes me is the sheer number of treatments that have been tried in rats and how little gets through to humans

    It's confusing to me but does it confuse you the scientists trying to sort out what might work and what might not?

    I urge the commencement of as human clinical trials of the most likely therapies so that eventually something will work.

  3. #13
    I just don't get it though with the Rat studies. In the past so much has worked with them that has been useless with humans. I remember Chris Reeve saying "Oh to be a Rat" So at this point I really can't get excited at all with Rat studies with all the things I've seen since the swedish study in 1996 produced some real positive results using Rats, I had my hopes up for sure I was going to walk again even back then.

    Everybody should read the link I posted, it is very interesting and informative even though it was written a while back. It's just goes to show how complex all this stuff it and how we are still even now learning so much about the Spinal Cord.
    Last edited by Curt Leatherbee; 12-08-2011 at 08:17 PM.
    "Life is about how you
    respond to not only the
    challenges you're dealt but
    the challenges you seek...If
    you have no goals, no
    mountains to climb, your
    soul dies".~Liz Fordred

  4. #14
    Quote Originally Posted by Curt Leatherbee View Post
    I just don't get it though with the Rat studies. In the past so much has worked with them that has been useless with humans. I remember Chris Reeve saying "Oh to be a Rat" So at this point I really can't get excited at all with Rat studies with all the things I've seen since the swedish study in 1996 produced some real positive results using Rats, I had my hopes up for sure I was going to walk again even back then.
    Curt, without animal studies, we would have nothing! They serve as a guide for therapies that we take to clinical trials. People cannot serve as guinea pigs. Very few therapies that have worked in rats have been taken to clinical trial. The reason why the therapies have not been taken to clinical trial has nothing to do with rats being a bad model for spinal cord injury but lack of money for clinical trials. Don't blame the rat for the failure of funding.

    What are the therapies that have resulted in some functional recovery of rats after well-defined spinal cord injuries? Take a look at some therapies that have been reported to work in rats, published in this paper.

    How many of these therapies that reported to work in rats have ever been taken to clinical trial? Before you blame rat spinal cord injury, think about the real reasons why the therapies have not gone to trial. Part of it is the back-biting that is going on, the paucity of funding for clinical trials, and the nihilism in our field.


  5. #15
    None of those reasons is good enough

    If by nihilism you mean the researchers still don't believe in a cure then they are surely in the wrong field - even I as a complete layman can see that in principal sci is curable

  6. #16
    I understand what you are saying Wise, it is all just so frusterating. I wish I were strong enough to organize things from the political level. Years ago I really felt that a major revolution might be the answer. I guess I had those thoughts from a class I took in High School called "Revolution and Reaction" I even remember the name of the teacher, dr. Bressler. By revolution I mean like big groups of Spinal Cord Injured people protesting right in front of the White house and being caught on camera being dragged away by MP's, I'll bet the news groups definately would have been there then to catch that and it would have infurated the public. I really almost feel though now it is too late for that. I am just kinda shocked that the general public was not more demanding of the government towards NIH involvement in regenerative medicine research. They will more than likely all be affected in one form or another if they live long enough. The ability to regrow ones own organs for transplants, regrow central nerve cells, etc would be so huge and such a big industry and job provider for the USA. Why was not there ever any political initiative to do this? I know Kerry had talked about it some. I remember talking to him some about it when I bumped into him in the walkway under I think it was the Russell bldg. back in around 2003. We really definatly need some kind of leader, especially after the passing of Chris Reeve.
    "Life is about how you
    respond to not only the
    challenges you're dealt but
    the challenges you seek...If
    you have no goals, no
    mountains to climb, your
    soul dies".~Liz Fordred

  7. #17
    I would just like to say that every paper that makes specific findings is an extremely important puzzle piece not just for spinal cord injury but for regenerative Medicine itself . " The fact that they don’t express HLA-DR is of interest because this is one of the important tissue compatibility antigens that lead to immune rejection if not matched. Finally, these DPDS cells express nestin (a neural stem cell marker) and other markers of differentiated neural cells including GFAP (for differentiated astrocytes), beta-III tubulin and NeuN (for differentiated neurons), A2B5 (a glial-restricted precursor marker), and MBP (or myelin basic protein which is associated with myelin). So, these cells from teeth express markers of neural stem cells.
    " Could lead to a large pool of mass produce stem cells That could be universally transplanted Which would be much cheaper And safer.

    There are a lot of Legitimate treatments that can go to human trials . The cost is enormous and each individual trial could lead to more research and more trials With a small amount of hope for an immediate financial payoff . I would like to wake up one day and here that the president Is going to cut one trillion dollars over the next 10 years from the military budget and funnel that money for education , medical research And human trials for regenerative medicine. But before I was injured My next door neighbor's son suffered a spinal cord injury And was able to walk with crutches. I never really knew the nature of his injury. I am ashamed of myself for walking through life with blinders on never noticing the pain and suffering that exists in the world but for few dollars could be wiped out.

  8. #18
    n my opinion, the authors have taken one of the most severe spinal cord injury models and showed that transplantation of two types of cells result in evidence of regeneration and recovery of hindlimb movements. The histological and behavioral evidence support each other. The type of cells that produce less histological improvement stimulated less recovery. The authors showed that the SHED cells seem to support axonal growth in culture better and this is interesting as well. To me, this paper deserves publication. It is all right if some people decide that they are not convinced but the paper needs to see the light of day instead of being buried. This debate is good. To tell you the truth, I don’t think that the data for chondroitinase efficacy for spinal cord injury is that much better.

    To Wise from Jerry.

    Frankly, I am not surprised that you yet again reveal your complete lack of ability to critically evaluate the merits of the published literature. I neither have the time nor the desire to critique the list of minimally interesting (and indeed mostly garbage) papers that you spent much too much time gathering. I have far more important things to do. One thing that I will NEVER do (and should be deemed at a minimum to be unethical if not criminal) is to spread false hope to the SCI community in order to raise money. Would you agree?

  9. #19
    Who is speading false hope to raise money? I know one issue, that is unethical if not criminal, is competing for recognition, money and accolades instead of cooperating to find an answer for this eternal living hell called sci.

  10. #20
    From what I have critically evaluated. the answer lies in a pten inhibitor that can be delivered and localized. NOTHING ELSE ALLOWS ROBUST Corticospinal Neuronal growth.

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