A dermal niche for multipotent adult skin-derived precursor cells

A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.     

Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny

This protocol describes methods of isolating skin-derived precursors (SKPs) from rodent and human skin, and for generating and enriching Schwann cells from rodent SKPs. SKPs are isolated as a population of non-adherent cells from the dermis that proliferate and self-renew as floating spheres in response to fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). Their differentiation into Schwann cells and subsequent enrichment of these differentiated progeny involves culturing SKPs as adherent cells in the absence of FGF2 and EGF, but in the presence of neuregulins, and then mechanically isolating the Schwann cell colonies using cloning cylinders. Methods for expanding and characterizing these Schwann cells are provided. Generation of primary SKPs takes approximately 2 weeks, while differentiation of Schwann cells requires an additional 4-6 weeks.     

Isolation of multipotent adult stem cells from the dermis of mammalian skin

We describe here the isolation of stem cells from juvenile and adult rodent skin. These cells derive from the dermis, and clones of individual cells can proliferate and differentiate in culture to produce neurons, glia, smooth muscle cells and adipocytes. Similar precursors that produce neuron-specific proteins upon differentiation can be isolated from adult human scalp. Because these cells (termed SKPs for skin-derived precursors) generate both neural and mesodermal progeny, we propose that they represent a novel multipotent adult stem cell and suggest that skin may provide an accessible, autologous source of stem cells for transplantation.     

Characterization of multipotent adult stem cells from the skin: transforming growth factor-beta (TGF-beta) facilitates cell growth

Recently, adult stem cells have been isolated from the skin and designated as skin-derived precursors (SKPs). These SKPs, cultured in vitro, can give rise to neurons, glia, smooth muscle cells, and adipocytes. In the current study, we confirmed the clonal expansion of SKPs using a sphere-forming culture system in a medium containing methylcellulose. Among the growth factors, only transforming growth factor-beta (TGF-beta) was revealed to uniquely facilitate the sphere formation and proliferation of the SKPs in combination with EGF and bFGF. In addition, TGF-beta did not alter phenotypical characteristics of the SKPs under sphere-forming conditions. The effect of TGF-beta on sphere formation was not observed in neural stem cells, which expressed a different set of cell surface markers from SKPs, suggesting that SKPs have distinct features. Although the number of SKPs decreased with age, TGF-beta increased the sphere colony formation and proliferation in all ages. These results suggest that SKPs maintained in the presence of TGF-beta during culture are of potential use in cell-replacement therapies employing adult tissue sources.     

Isolation and Characterization of Two Kinds of Stem Cells from the Same Human Skin Back Sample with Therapeutic Potential in Spinal Cord Injury

Backgrounds and Objective

Spinal cord injury remains to be a challenge to clinicians and it is attractive to employ autologous adult stem cell transplantation in its treatment, however, how to harvest cells with therapeutic potential easily and how to get enough number of cells for transplantation are challenging issues. In the present study, we aimed to isolate skin-derived precursors (SKPs) and dermal multipotent stem cells (dMSCs) simultaneously from single human skin samples from patients with paraplegia.

Methods

Dissociated cells were initially generated from the dermal layer of skin samples from patients with paraplegia and cultured in SKPs proliferation medium. Four hours later, many cells adhered to the base of the flask. The suspended cells were then transferred to another flask for further culture as SKPs, while the adherent cells were cultured in dMSCs proliferation medium. Twenty-four hours later, the adherent cells were harvested and single-cell colonies were generated using serial dilution method. [³H]thymidine incorporation assay, microchemotaxis Transwell chambers assay, RT-PCR and fluorescent immunocytochemistry were employed to examine the characterizations of the isolated cells.

Results

SKPs and dMSCs were isolated simultaneously from a single skin sample. SKPs and dMSCs differed in several respects, including in terms of intermediate protein expression, proliferation capacities, and differentiation tendencies towards mesodermal and neural progenies. However, both SKPs and dMSCs showed high rates of differentiation into neurons and Schwann cells under appropriate inducing conditions. dMSCs isolated by this method showed no overt differences from dMSCs isolated by routine methods.

Conclusions

Two kinds of stem cells, namely SKPs and dMSCs, can be isolated simultaneously from individual human skin sample from paraplegia patients. Both of them show ability to differentiate into neural cells under proper inducing conditions, indicating their potential for the treatment of spinal cord injury patients by autologous cell transplantation.     

The PI3K-Akt pathway inhibits senescence and promotes self-renewal of human skin-derived precursors in vitro

Skin-derived precursors (SKPs) are embryonic neural crest- or somite-derived multipotent progenitor cells with properties of dermal stem cells. Although a large number of studies deal with their differentiation ability and potential applications in tissue damage repair, only a few studies have concentrated on the regulation of SKP self-renewal. Here, we found that after separation from their physiological microenvironment, human foreskin-derived SKPs (hSKPs) quickly senesced and lost their self-renewal ability. We observed a sharp decrease in Akt activity during this process, suggesting a possible role of the PI3K-Akt pathway in hSKP maintenance in vitro. Blocking this pathway with several inhibitors inhibited hSKP proliferation and sphere formation and increased hSKP senescence. In contrast, activating this pathway with PDGF-AA and a PTEN inhibitor, bpV(pic), promoted proliferation, improved sphere formation, and alleviated senescence of hSKPs, without altering their differentiation potential. Data also implied that this effect was associated with altered actions of FoxO3 and GSK-3β. Our results suggest an important role of the PI3K-Akt pathway in the senescence and self-renewal of hSKPs. These findings also provide a better understanding of the cellular mechanisms underlying hSKP self-renewal and stem cell senescence to allow more efficient expansion of hSKPs for regenerative medical applications.     

Migration and differentiation of Langerhans cell precursors

Epidermal Langerhans cells (LC) are the first sentinels of the skin immune system. To study immigration of human LC precursor cells into the skin, we established a two-compartmental skin model consisting of a dermal matrix and an epidermal sheet of keratinocytes. We tested the individual components of the skin model for their influence on phenotype and function of LC precursors. A time window at day 5/6 of differentiation was determined, during which in vitro generated LC precursors expressed adhesion molecules and chemokine receptors required for transmigration across endothelial cell layers and the dermis towards the epidermis. They expressed L-selectin, integrins, platelet endothelial cell adhesion molecule-1, E-cadherin and CC-chemokine receptor 6 and were thus fitted out for transendothelial migration and immigration into the dermis. In a transwell system, these LC precursors migrated towards the chemokine MIP3alpha, demonstrating functional integrity of chemokine receptor 6. For the in vitro reconstituted skin, keratinocytes were grown on a de-epidermized dermis for one to three weeks and formed an epidermal sheet. We allowed LC precursor cells to migrate into this two-compartmental model from the dermal side and examined the presence of CD1alpha--positive cells. LC precursors migrated through the dermal matrix towards the layer of keratinocytes representing the epidermis and could be identified by immunohistology. Experiments designed to investigate the influence of signals provided by both the skin components and by the LC precursors on LC immigration into the skin are in progress.     

Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.     

Generation of cerebellar neuron precursors from embryonic stem cells

Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.     

Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas

During pancreatic development, endocrine and exocrine cell types arise from common precursors in foregut endoderm. However, little information is available regarding regulation of pancreatic epithelial differentiation in specific precursor populations. We show that undifferentiated epithelial precursors in E10.5 mouse pancreas express nestin, an intermediate filament also expressed in neural stem cells. Within developing pancreatic epithelium, nestin is co-expressed with pdx1 and p48, but not ngn3. Epithelial nestin expression is extinguished upon differentiation of endocrine and exocrine cell types, and no nestin-positive epithelial cells are observed by E15.5. In E10.5 dorsal bud explants, activation of EGF signaling results in maintenance of undifferentiated nestin-positive precursors at the expense of differentiated acinar cells, suggesting a precursor/progeny relationship between these cell types. This relationship was confirmed by rigorous lineage tracing studies using nestin regulatory elements to drive Cre-mediated labeling of nestin-positive precursor cells and their progeny. These experiments demonstrate that a nestin promoter/enhancer element containing the second intron of the mouse nestin locus is active in undifferentiated E10.5 pancreatic epithelial cells, and that these nestin-positive precursors contribute to the generation of differentiated acinar cells. As in neural tissue, nestin-positive cells act as epithelial progenitors during pancreatic development, and may be regulated by EGF receptor activity.     

Cell interactions within nascent neural crest cell populations transiently promote death of neurogenic precursors

We have previously shown that cultured trunk neural crest cell populations irreversibly lose neurogenic ability when dispersal is prevented or delayed, while the ability to produce other crest derivatives is retained (Vogel, K. S. and Weston, J. A. (1988) Neuron 1, 569-577). Here, we show that when crest cells are prevented from dispersing, cell death is increased and neurogenesis is decreased in the population, as a result of high cell density. Control experiments to characterize the effects of high cell density on environmental conditions in culture suggest that reduced neurogenesis is the result of cell-cell interactions and not changes (conditioning or depletion) of the culture medium. Additionally, we show that the caspase inhibitor zVAD-fmk, which blocks developmentally regulated cell death, rescues the neurogenic ability of high density cultures, without any apparent effect on normal, low-density cultures. We conclude, therefore, that increased cell interaction at high cell densities results in the selective death of neurogenic precursors in the nascent crest population. Furthermore, we show that neurogenic cells in cultured crest cell populations that have dispersed immediately are not susceptible to contact-mediated death, even if they are subsequently cultured at high cell density. Since most early migrating avian crest cells express Notch1, and a subset expresses Delta1 (Wakamatsu, Y., Maynard, T. M. and Weston, J. A. (2000) Development 127, 2811-2821), we tested the possibility that the effects of cell contact were mediated by components of a Notch signaling pathway. We found that neurogenic precursors are eliminated when crest cells are co-cultured with exogenous Delta1-expressing cells immediately after they segregate from the neural tube, although not after they have previously dispersed. We conclude that early and prolonged cell interactions, mediated at least in part by Notch signaling, can regulate the survival of neurogenic cells within the nascent crest population. We suggest that a transient episode of cell contact-mediated death of neurogenic cells may serve to eliminate fate-restricted neurogenic cells that fail to disperse promptly in vivo.     

Brain engraftment and therapeutic potential of stem/progenitor cells derived from mouse skin

Skin stem/progenitor cells (SKPs) derive from the dermis and in culture can generate mesodermal and neural progenies. To investigate their potential for the treatment of brain diseases, we first injected SKPs into the brain of syngeneic mice. Brain histology indicated that most SKPs remained undifferentiated and clustered at the injection site, while, in vitro, 17% of SKPs expressed neural markers, as assessed by flow cytometry. After labeling with magnetodendrimers, murine and human SKPs were detected by magnetic resonance imaging even 5 months after brain injection. To evaluate their therapeutic potential on malignant gliomas, IL-4 SKPs (i.e. SKPs transduced by a lentiviral vector carrying the cDNA of the anti-glioma cytokine interleukin-4) were injected into GL261 experimental gliomas. IL-4-SKPs prolonged significantly the survival of tumor-bearing mice: furthermore, GL261 gliomas attracted SKPs originally injected into the contralateral hemisphere. Thus, prolonged survival, capacity for transgene expression, and lack of uncontrolled proliferation suggest that SKPs warrant further consideration as therapeutic tools for brain tumors and, possibly, other neurological disorders.     

Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos

Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. Here, we have uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. We first demonstrate that the segregation of notochord and neural fates is an autonomous property of the mother cell and that the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. We show that these cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. Finally, we demonstrate that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. We propose a model whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification.     

Neuronal differentiation from postmitotic precursors in the ciliary ganglion

In the chick ciliary ganglion, neuronal number is kept constant between St. 29 and St. 34 (E6-E8) despite a large amount of cell death. Here, we characterize the source of neurogenic cells in the ganglion as undifferentiated neural crest-derived cells. At St. 29, neurons and nonneuronal cells in the ciliary ganglion expressed the neural crest markers HNK-1 and p75(NTR). Over 50% of the cells were neurons at St. 29; of the nonneuronal cells, a small population expressed glial markers, whereas the majority was undifferentiated. When placed in culture, nonneuronal cells acquired immunoreactivity for HuD, suggesting that they had commenced neuronal differentiation. The newly differentiated neurons arose from precursors that did not incorporate bromodeoxyuridine. To test whether these precursors could undergo neural differentiation in vivo, purified nonneuronal cells from St. 29 quail ganglia were transplanted into chick embryos at St. 9-14. Subsequently, quail cells expressing neuronal markers were found in the chick ciliary ganglion. The existence of this precursor pool was transient because nonneuronal cells isolated from St. 38 ganglia failed to form neurons. Since all ciliary ganglion neurons are born prior to St. 29, these results demonstrate that there are postmitotic neural crest-derived precursors in the developing ciliary ganglion that can differentiate into neurons in the appropriate environment.     

Neural precursors express multiple chondroitin sulfate proteoglycans, including the lectican family

Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid (CSF) of both human neonates with preterm hydrocephalus, and P8 hydrocephalic mice. We hypothesized CSF CSPGs are synthesized by neural precursors, separated from ventricular CSF by ependyma, which is often disrupted in hydrocephalus. Western blotting demonstrates that neural precursors cultured as neurospheres secrete CSPGs (> 30 microg/ml) into their media which appear to be very similar to these CSF CSPGs. Some CSPGs bear the stage-specific embryonic antigen-1 (ssea-1), associated with embryonic/neural stem cells. Neurospheres transcribe many CSPG genes, including the entire aggrecan/lectican family, phosphacan, and tenascin. Phosphacan can be detected in media by Western blotting. Aggrecan can be detected in media after purification using hyaluronic acid affinity chromatography. During differentiation, neurospheres downregulate CSPGs. This is the first report to show that proliferating neural precursors synthesize lecticans, including aggrecan, which are downregulated with differentiation. These observations suggest novel links between CSPGs and CNS precursor biology.