Neural crest stem cell property of apical pulp cells derived from human developing tooth

Recent reports have described that NCSCs (neural crest-derived stem cells) are not only present in the embryonic neural crest but also in the adult tissues. Dental pulp is one of mesenchymal soft tissues origin from cranial neural crest cells, and thought to be a source of adult stem cells. Here, we investigated the existence of NCSC-like cells in apical pulp of human developing tooth. Human impacted third molars with immature apex freshly extracted were obtained. The cells derived from the apical pulp tissue not framed by dentin or the coronal pulp tissues were cultured by primary explant culture. APDCs (apical pulp-derived cells) and CPCs (coronal pulp cells) formed spheres under neurosphere culture condition. The number of spheres from APDCs was larger than that from CPCs. The sphere-forming cells derived from APDCs had self-renewal capacity, and expressed neural crest-associated markers (p75, Snail and Slug) and NSC (neural stem cell) markers (Nestin and Musashi1). The expression pattern of mesenchymal stem cell markers, CD105 and CD166, on the surface of sphere-forming cells derived APDCs was different from that of APDCs. These sphere-forming cells could differentiate into multiple mesenchymal lineages (osteoblasts, adipocytes, chondrocytes and smooth muscle cells) and neural lineage (neurons) in vitro, and generated ectopic bone tissues on the border of HA (hydroxyapatite) scaffold in vivo. The results of this study suggest that APDCs contain cells with characteristics of NCSCs reported previously in mice. Humans developing tooth with immature apex is an effective source of cells for neural crest lineage tissue regeneration.     

Common precursors for neural and mesectodermal derivatives in the cephalic neural crest.

The cephalic neural crest (NC) of vertebrate embryos yields a variety of cell types belonging to the neuronal, glial, melanocytic and mesectodermal lineages. Using clonal cultures of quail migrating cephalic NC cells, we demonstrated that neurons and glial cells of the peripheral nervous system can originate from the same progenitors as cartilage, one of the mesectodermal derivatives of the NC. Moreover, we obtained evidence that the migrating cephalic NC contains a few highly multipotent precursors that are common to neurons, glia, cartilage and pigment cells and which we interprete as representative of a stem cell population. In contrast, other NC cells, although provided with identical culture conditions, give rise to clones composed of only one or some of these cell types. These cells thus appear restricted in their developmental potentialities compared to multipotent cells. It is therefore proposed that, in vivo, the active proliferation of pluripotent NC cells during the migration process generates distinct subpopulations of cells that become progressively committed to different developmental fates.     

Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine.     

Neural crest development is regulated by the transcription factor Sox9

The neural crest is a transient migratory population of stem cells derived from the dorsal neural folds at the border between neural and non-neural ectoderm. Following induction, prospective neural crest cells are segregated within the neuroepithelium and then delaminate from the neural tube and migrate into the periphery, where they generate multiple differentiated cell types. The intrinsic determinants that direct this process are not well defined. Group E Sox genes (Sox8, Sox9 and Sox10) are expressed in the prospective neural crest and Sox9 expression precedes expression of premigratory neural crest markers. Here, we show that group E Sox genes act at two distinct steps in neural crest differentiation. Forced expression of Sox9 promotes neural-crest-like properties in neural tube progenitors at the expense of central nervous system neuronal differentiation. Subsequently, in migratory neural crest cells, SoxE gene expression biases cells towards glial cell and melanocyte fate, and away from neuronal lineages. Although SoxE genes are sufficient to initiate neural crest development they do not efficiently induce the delamination of ectopic neural crest cells from the neural tube consistent with the idea that this event is independently controlled. Together, these data identify a role for group E Sox genes in the initiation of neural crest development and later SoxE genes influence the differentiation pathway adopted by migrating neural crest cells.     

The adult hair follicle: cradle for pluripotent neural crest stem cells

This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.     

Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose‐derived stem cells: Comparison with fetal chondrocytes

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose‐derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven‐mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF‐β1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4‐ and 6.1‐fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF‐β1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530‐fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold‐based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose‐derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393–401. © 2010 Wiley Periodicals, Inc.     

EGF–FGF2 stimulates the proliferation and improves the neuronal commitment of mouse epidermal neural crest stem cells (EPI-NCSCs)

Epidermal neural crest stem cells (EPI-NCSCs), which reside in the bulge of hair follicles, are attractive candidates for several applications in cell therapy, drug screening and tissue engineering. As suggested remnants of the embryonic neural crest (NC) in an adult location, EPI-NCSCs are able to generate a wide variety of cell types and are readily accessible by a minimally invasive procedure. Since the combination of epidermal growth factor (EGF) and fibroblast growth factor type 2 (FGF₂) is mitogenic and promotes the neuronal commitment of various stem cell populations, we examined its effects in the proliferation and neuronal potential of mouse EPI-NCSCs. By using a recognized culture protocol of bulge whiskers follicles, we were able to isolate a population of EPI-NCSCs, characterized by the migratory potential, cell morphology and expression of phenotypic markers of NC cells. EPI-NCSCs expressed neuronal, glial and smooth muscle markers and exhibited the NC-like fibroblastic morphology. The treatment with the combination EGF and FGF₂, however, increased their proliferation rate and promoted the acquisition of a neuronal-like morphology accompanied by reorganization of neural cytoskeletal proteins βIII-tubulin and nestin, as well as upregulation of the pan neuronal marker βIII-tubulin and down regulation of the undifferentiated NC, glial and smooth muscle cell markers. Moreover, the treatment enhanced the response of EPI-NCSCs to neurogenic stimulation, as evidenced by induction of GAP43, and increased expression of Mash-1 in neuron-like cell, both neuronal-specific proteins. Together, the results suggest that the combination of EGF–FGF2 stimulates the proliferation and improves the neuronal potential of EPI-NCSCs similarly to embryonic NC cells, ES cells and neural progenitor/stem cells of the central nervous system and highlights the advantage of using EGF–FGF₂ in neuronal differentiation protocols.     

Neuronal-glial interactions in central nervous system neurogenesis: the neural stem cell perspective

Essentially, three neuroectodermal-derived cell types make up the complex architecture of the adult CNS: neurons, astrocytes and oligodendrocytes. These elements are endowed with remarkable morphological, molecular and functional heterogeneity that reaches its maximal expression during development when stem/progenitor cells undergo progressive changes that drive them to a fully differentiated state. During this period the transient expression of molecular markers hampers precise identification of cell categories, even in neuronal and glial domains. These issues of developmental biology are recapitulated partially during the neurogenic processes that persist in discrete regions of the adult brain. The recent hypothesis that adult neural stem cells (NSCs) show a glial identity and derive directly from radial glia raises questions concerning the neuronal-glial relationships during pre- and post-natal brain development. The fact that NSCs isolated in vitro differentiate mainly into astrocytes, whereas in vivo they produce mainly neurons highlights the importance of epigenetic signals in the neurogenic niches, where glial cells and neurons exert mutual influences. Unravelling the mechanisms that underlie NSC plasticity in vivo and in vitro is crucial to understanding adult neurogenesis and exploiting this physiological process for brain repair. In this review we address the issues of neuronal/glial cell identity and neuronal-glial interactions in the context of NSC biology and NSC-driven neurogenesis during development and adulthood in vivo, focusing mainly on the CNS. We also discuss the peculiarities of neuronal-glial relationships for NSCs and their progeny in the context of in vitro systems.     

Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function

Neural crest progenitor cells are the main contributors to craniofacial cartilage and connective tissue of the vertebrate head. These progenitor cells also give rise to the pigment, neuronal and glial cell lineages. To study the molecular basis of neural crest differentiation, we have cloned the gene disrupted in the mont blanc (mob(m610)) mutation, which affects all neural crest derivatives. Using a positional candidate cloning approach we identified an A to G transition within the 3' splice site of the sixth intron of the tfap2a gene that abolishes the last exon encoding the crucial protein dimerization and DNA-binding domains. Neural crest induction and specification are not hindered in mob(m610) mutant embryos, as revealed by normal expression of early neural crest specific genes such as snail2, foxd3 and sox10. In addition, the initial stages of cranial neural crest migration appear undisturbed, while at a later phase the craniofacial primordia in pharyngeal arches two to seven fail to express their typical set of genes (sox9a, wnt5a, dlx2, hoxa2/b2). In mob(m610) mutant embryos, the cell number of neuronal and glial derivatives of neural crest is greatly reduced, suggesting that tfap2a is required for their normal development. By tracing the fate of neural crest progenitors in live mont blanc (mob(m610)) embryos, we found that at 24 hpf neural crest cells migrate normally in the first pharyngeal arch while the preotic and postotic neural crest cells begin migration but fail to descend to the pharyngeal region of the head. TUNEL assay and Acridine Orange staining revealed that in the absence of tfap2a a subset of neural crest cells are unable to undergo terminal differentiation and die by apoptosis. Furthermore, surviving neural crest cells in tfap2a/mob(m610) mutant embryos proliferate normally and later differentiate to individual derivatives. Our results indicate that tfap2a is essential to turn on the normal developmental program in arches 2-7 and in trunk neural crest. Thus, tfap2a does not appear to be involved in early specification and cell proliferation of neural crest, but it is a key regulator of an early differentiation phase and is required for cell survival in neural crest derived cell lineages.     

The Effect of Dexamethasone and Triiodothyronine on Terminal Differentiation of Primary Bovine Chondrocytes and Chondrogenically Differentiated Mesenchymal Stem Cells

The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal differentiation, they can be used to generate a model of endochondral ossification, but this limitation must be kept in mind when using them in cartilage tissue engineering application.     

Epigenetic modifiers promote efficient generation of neural-like cells from bone marrow-derived mesenchymal cells grown in neural environment

Understanding mechanisms that govern cell fate decisions will lead to developing techniques for induction of adult stem cell differentiation to desired cell outcomes and, thus, production of an autologos source of cells for regenerative medicine. Recently, we demonstrated that stem cells derived from adult central nervous system or bone marrow grown with other cell lineages or with more undifferentiated cells sometimes take on those characteristics. This indicates that manipulating extracellular factors may be sufficient to alter some developmental restrictions regulated by the epigenetic system. In this study, using pharmacological agents that interfere with the main components of the epigenetic program such as DNA methylation and histone deacetylation, we induce high-level expression of embryonic and neural stem cell (NSC) marker Sox2 in bone marrow-derived mesenchymal stem cells (MSCs). Exposure of these modified cells to a neural environment via juxtacrine and paracrine interactions promote efficient generation of neural stem-like cells as well as cells with neuronal and glial characteristics. We concluded that the manipulation strategy used in this study can be a useful method for efficient production of NSC-like cells from MSCs.     

Isolation and characterization of neural crest-derived stem cells from dental pulp of neonatal mice

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.     

Steroid-mediated differentiation of neural/neuronal cells from epithelial ovarian precursors in vitro

We reported earlier that occasional neurons evolve in human cultures of pluripotent ovarian epithelial stem cells. In subsequent experiments, frequent transdifferentiation into neural stem cells (NSC) and differentiating neurons was observed in human ovarian epithelial stem cells and porcine granulosa cells after exposure to certain combinations of sex steroids. Testosterone (TS), progesterone (PG) or estradiol (E2) alone do not increase the emergence of neurons. However, a mixture of TS+PG after E2 pretreatment converted a majority of ovarian epithelial stem cells or porcine granulosa cells into NSC and differentiating neuronal cells within one to three hours. Cultured neurons manifested an interconnectivity resembling primitive neuronal pathways in culture. These converted cells expressed the cell markers SSEA-1, SSEA-4, NCAM, and Thy-1 glycoconjugates of NSC and neurons, and differentiating cells showed characteristic neuronal morphology. Emergence of NSC and neuronal cells was associated with significant cellular depletion of L-glutamic acid (glutamate), which serves as the major excitatory neurotransmitter in the vertebrate CNS and its fast removal is essential for preventing glutamate excitotoxicity. These observations suggest that certain sequential systemic treatment with common sex steroids and their mixture might be effective in the treatment or prevention of degenerative CNS disorders. The ovarian stem cell cultures readily obtainable from human ovaries regardless of the woman's age have the potential to produce NSC for autologous regenerative treatment of neurologic diseases in aging women. Finally, the proper combination of sex steroids could possibly be employed for transdifferentiation of adult bone marrow stem cells or mobilized peripheral blood cells into autologous NSC and stimulate their neuronal differentiation after homing in the CNS.     

Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system

The discovery of neural stem cells (NSC) which ensure continuous neurogenesis in the adult mammalian brain, has led to a conceptual revolution in basic neuroscience and to high hopes for clinical nervous tissue repair. However, several research issues remain to address before neural stem cells can be harnessed for regenerative therapies. The presence of NSC in a nervous structure is demonstrated in vitro by primary culture of dissociated adult nervous tissue in the presence of the specific mitogens EGF and bFGF. This leads to spherical masses of proliferating cells endowed with capacities for self-renewal and, after growth factor removal, differentiation into the three characteristic cell types of nervous tissue (neurons, astrocytes, oligodendrocytes). In vivo, neurogenesis per se, i.e. production of new neurons, occurs only in a small subset of NSC-endowed structures. The production of oligodendrocytes, i.e. myelinating glial cells, is similarly restricted. Such in vivo restrictions were formally demonstrated to arise from the tissular microenvironnement, which led to the emerging concept of "neurogenic niche". In this context, major challenges now consist in identifying the nature of tissue-specific extracellular signals that determine lineage commitment of NSC progeny, understanding why NSCs display weak in vivo reactivity to lesions compared to other stem cell types in adults, and identifying the factors behind the very high resistance to tumorigenesis displayed by NSCs. Altogether, the current data offer hope for the future use of adult NSCs in regenerative therapies, provided that tissue-specific signals are identified in view of counteracting the intrinsic repression of new cell genesis and/or stimulating endogenous NSC recruitment to lesion sites.     

Induction and prevention of chondrocyte hypertrophy in culture

Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.     

Glycogen Synthase Kinase-3β Regulates Equilibrium Between Neurogenesis and Gliogenesis in Rat Model of Parkinson’s Disease: a Crosstalk with Wnt and Notch Signaling

Neurogenesis involves generation of functional newborn neurons from neural stem cells (NSCs). Insufficient formation or accelerated degeneration of newborn neurons may contribute to the severity of motor/nonmotor symptoms of Parkinson’s disease (PD). However, the functional role of adult neurogenesis in PD is yet not explored and whether glycogen synthase kinase-3β (GSK-3β) affects multiple steps of adult neurogenesis in PD is still unknown. We investigated the possible underlying molecular mechanism of impaired adult neurogenesis associated with PD. Herein, we show that single intra-medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA) efficiently induced long-term activation of GSK-3β and reduced NSC self-renewal, proliferation, neuronal migration, and neuronal differentiation accompanied with increased astrogenesis in subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Indeed, 6-OHDA also delayed maturation of neuroblasts in the DG as witnessed by their reduced dendritic length and arborization. Using a pharmacological approach to inhibit GSK-3β activation by specific inhibitor SB216763, we show that GSK-3β inhibition enhances radial glial cells, NSC proliferation, self-renewal in the SVZ, and the subgranular zone (SGZ) in the rat PD model. Pharmacological inhibition of GSK-3β activity enhances neuroblast population in SVZ and SGZ and promotes migration of neuroblasts towards the rostral migratory stream and lesioned striatum from dorsal SVZ and lateral SVZ, respectively, in PD model. GSK-3β inhibition enhances dendritic arborization and survival of granular neurons and stimulates NSC differentiation towards the neuronal phenotype in DG of PD model. The aforementioned effects of GSK-3β involve a crosstalk between Wnt/β-catenin and Notch signaling pathways that are known to regulate NSC dynamics.     

Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2

BACKGROUND: Adipose tissue has been demonstrated to contain a population of progenitor cells that can differentiate into bone and cartilage. Studies have suggested that adipose-derived adult stem (ADAS) cells can be induced to differentiate into chondrocytes by transforming growth factor-beta (TGF-beta). In this study, we examined whether bone morphogenetic protein-2 (BMP-2), as a member of the TGF-beta superfamily, could regulate ADAS cells to differentiate into a chondrolineage. METHODS: ADAS cells were isolated and induced by rhBMP-2. These cells were cultured in pellets for 2 weeks, and the chondrogenic phenotype was observed in vitro and in vivo. ADAS cells cultured without BMP-2 were used as controls. RESULTS: After 2 weeks of culture, the differentiated ADAS cells reacted positively to Alcian blue and collagen II, and the content of collagen II protein was obviously up-regulated at day 14. Glycosaminoglycan (GAG) content gradually increased from day 2 to day 14 (P < 0.05). However, H&E staining and collagen II expression were weak, and there was a little collagen II protein and GAG detected in the control group. Additionally, the pellets of ADAS cells induced by rhBMP-2 were transplanted into BALB/C nude mice and formed cartilage lacuna at week 8 in vivo. DISCUSSION: These data demonstrate that rhBMP-2 induce ADAS cells to differentiate into chondrocytes in vitro and in vivo. This is useful for basic and clinical studies aimed at repairing cartilage damage. But in a control group, ADAS cells tended towards differentiation into chondrocytes, which was affected by ITS. We will be exploring the mechanism further.     

Development of mesenchymal stem cells partially originate from the neural crest

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells isolated from many adult tissues. Previous studies reported that MSCs can differentiate to both mesodermal and neural lineages by a phenomenon referred to as ‘‘dedifferentiation’’ or ‘‘transdifferentiation’’. However, since MSCs have only been defined in vitro, much of their development in vivo is still unknown. Here, we prospectively identified MSCs in the bone marrow from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. EGFP-positive MSCs formed spheres that expressed neural crest stem cell genes and differentiated into neurons, glial cells, and myofibroblasts. Interestingly, we observed MSCs both in the GFP⁺ and GFP⁻ fraction and found that there were no significant differences in the in vitro characteristics between these two populations. Our results suggest that MSCs in adult bone marrow have at least two developmental origins, one of which is the neural crest.