Human Skin-derived Mesenchymal Stem Cells Research Articles

1427 Induction of human skin-derived mesenchymal stem cells for the treatment of a Parkinsonism mouse model

1427 Induction of human skin-derived mesenchymal stem cells for the treatment of a Parkinsonism mouse model

Transplantation of Human Skin-Derived Mesenchymal Stromal Cells Improves Locomotor Recovery After Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a devastating neurologic disorder with significant impacts on quality of life, life expectancy, and economic burden. Although there are no fully restorative treatments yet available, several animal and small-scale clinical studies have highlighted the therapeutic potential of cellular interventions for SCI. Mesenchymal stem cells (MSCs)-which are conventionally isolated from the bone marrow-recently emerged as promising candidates for treating SCI and have been shown to provide trophic support, ameliorate inflammatory responses, and reduce cell death following the mechanical trauma. Here we evaluated the human skin as an alternative source of adult MSCs suitable for autologous cell transplantation strategies for SCI. We showed that human skin-derived MSCs (hSD-MSCs) express a range of neural markers under standard culture conditions and are able to survive and respond to neurogenic stimulation in vitro. In addition, using histological analysis and behavioral assessment, we demonstrated as a proof-of-principle that hSD-MSC transplantation reduces the severity of tissue loss and facilitates locomotor recovery in a rat model of SCI. Altogether, the study provides further characterization of skin-derived MSC cultures and indicates that the human skin may represent an attractive source for cell-based therapies for SCI and other neurological disorders. Further investigation is needed to elucidate the mechanisms by which hSD-MSCs elicit tissue repair and/or locomotor recovery.

Expression of neural markers by undifferentiated mesenchymal-like stem cells from different sources.

The spontaneous expression of neural markers, already demonstrated in bone marrow (BM) mesenchymal stem cells (MSCs), has been considered as evidence of the MSCs' predisposition to differentiate toward neural lineages, supporting their use in stem cell-based therapy for neural repair. In this study we have evaluated, by immunocytochemistry, immunoblotting, and flow cytometry experiments, the expression of neural markers in undifferentiated MSCs from different sources: human adipose stem cells (hASCs), human skin-derived mesenchymal stem cells (hS-MSCs), human periodontal ligament stem cells (hPDLSCs,) and human dental pulp stem cells (hDPSCs). Our results demonstrate that the neuronal markers βIII-tubulin and NeuN, unlike other evaluated markers, are spontaneously expressed by a very high percentage of undifferentiated hASCs, hS-MSCs, hPDLSCs, and hDPSCs. Conversely, the neural progenitor marker nestin is expressed only by a high percentage of undifferentiated hPDLSCs and hDPSCs. Our results suggest that the expression of βIII-tubulin and NeuN could be a common feature of stem cells and not exclusive to neuronal cells. This could result in a reassessment of the use of βIII-tubulin and NeuN as the only evidence proving neuronal differentiation. Further studies will be necessary to elucidate the relevance of the spontaneous expression of these markers in stem cells.

Fabrication and characterization of scaffold from cadaver goat-lung tissue for skin tissue engineering applications

The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H&E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications.

The usefulness of basic fibroblast growth factor for radiation‐exposed tissue

A high dose of ionizing external radiation damage to the skin and soft tissue results in changes in function as well as in the general body condition. Once radiation surpasses the tissue safety or survival level, progressive alteration in the damaged tissue results in tissue loss and then flap loss. Local expression and action of stem cells or local growth factors in the irradiated tissue is mitigated, and external administration is sought to investigate the possibility of skin and soft tissue survival after an elevating flap. Basic fibroblast growth factor (bFGF) is primarily considered as a potent angiogenic growth factor. In burns, resurfacing with a dermal component is required, and bFGF stimulates wound healing and enhances human skin-derived mesenchymal stem cells under serum-free conditions in a dose-dependent manner. Thirty-five male, 4- to 8-week-old CLAWN miniature pigs received radiation exposure to assess the effectiveness of bFGF in terms of the progressive clinical course relevant to human skin and soft tissue. At 2 weeks following 10-Gy irradiation, tissue was preserved in the group receiving subcutaneous placement of a round-type tissue expander and bFGF. The expander plus bFGF group demonstrated significantly greater dermo-epidermal proliferation than the radiation alone, radiation plus bFGF, or expander plus radiation plus vehicle-solution groups, and new blood vessel formation was significantly increased in the expander tissue with bFGF after irradiation (p < 0.01). Electron microscopy revealed that tissue with expander and bFGF maintained more stable skin adnexae with preserved intact epidermis and dermis. Thus, bFGF improved and maintained the tissue viability after immediate irradiation in the skin and soft tissue.

Culture-expanded human dermal stem cells exhibit donor to donor differences in cAMP generation

Stem cell techniques have facilitated a number of potential uses for such cells in cell therapy and drug development. Studies of the cAMP/protein kinase A (PKA) pathway are widely employed to investigate the effects of a large variety of substances. We assayed the cAMP pathway in human skin-derived mesenchymal stem cells (S-MSC) to evaluate donor to donor variations in response to pharmacological manipulations in vitro. Immunophenotyping of S-MSC revealed that, in general, 95% of S-MSCs were positive for CD90, CD73 and CD105 and negative for the expression of haemopoetic markers CD14, CD45 and human leukocyte antigen-DR (HLA-DR). Nevertheless, fluctuations occurred in basal cAMP levels from 5 pmol/mg to 18 pmol/mg. Total cAMP response element binding protein (CREB) concentrations ranged from 0.8ng/ml to 1ng/ml, whereas the proportions of phospho-CREB versus total CREB differed between the cell lines. Basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) stimulated cAMP generation, whereas leukaemia inhibiting factor reduced some of their effects. Forskolin (0.05 and 1mM) acted in synergy with FGF-2 and EGF; however, it caused pronounced donor to donor differences in the increase of cAMP and phospho-CREB levels. Additionally, dibutyryl-cAMP caused significant donor to donor variations in cell proliferation, possibly indicating a change of cell differentiation status. We speculate that similar donor diversity might be observed after cell stimulation with various G(s)-protein-coupled receptor ligands. Heterogeneity of donor cell responses to stimulation of the cAMP pathway indicates the need for wide safety margins for S-MSC use in drug screening; nevertheless, knowledge of this heterogeneity might be useful for the design of donor-specific cell therapy.

Biological safety of human skin-derived stem cells after long-term in vitro culture

The aim of this study was to investigate the biological safety of human skin-derived mesenchymal stem cells (HSMSCs) cultured in vitro by detecting changes in karyotype, major histocompatibility complex expression and tumorigenicity. Before the 21st passage of the in vitro culture, cell surface markers were analysed by flow cytometry; major histocompatibility complex expression was detected by RT-PCR and flow cytometry. The tumorigenicity of HSMSCs was tested using SCID mice and observing changes in the injection site and pathological sections. Flow cytometry demonstrated that HSMSCs express CD73, CD105 and vimentin, but haematopoietic markers CD34, CD45 and CD19 were not expressed. Human leukocyte antigen (HLA-I and HLA-DR) mRNA was detected by RT-PCR; the protein expression of HLA-I was 29.5-31.7%, but HLA-DR protein expression was not detected in HSMSCs. The result of karyotype analysis before the 21st passage was normal and tumour formation was not detected in the mice. Taken as a whole, our results suggest that HSMSCs cultured in vitro may be safely transplanted in vivo, due to moderate expression of HLA-I and low expression of HLA-DR, non-tumorigenicity and normal karyotype.

Skin‐derived mesenchymal stem cells (S‐MSCs) induce endothelial cell activation by paracrine mechanisms

The mesenchymal stem cells (MSCs) are able to accumulate at the site of tissue damage. For this reason, they must transmigrate across the endothelium. In this study, we focused on skin-derived MSCs (S-MSCs), because the skin represents a useful stem cell source, and we analysed the VEGF released by S-MSCs, because it is known to promote endothelial cell proliferation and vascular permeability. Moreover, we evaluated the influence of S-MSC-conditioned medium on human aortic endothelial cell intracellular calcium concentration ([Ca(2+)](i)) and nitric oxide (NO) production, given their important role in endothelial permeability modulation. Our results suggest that human S-MSCs may interact with the endothelium via paracrine mechanisms, probably leading to an alteration of the endothelial barrier. Consequently, we could hypothesize that a therapeutic approach based on human skin-derived MSCs may have a positive effect on tissue repair.

Human skin-derived mesenchymal stem cells as a source of VEGF and nitric oxide

Researches on stem cells bring promise to functional skin repair. In particular, it has been recently suggested that mesenchymal stem cells (MSCs) could positively affect cutaneous wound healing through differentiation and paracrine action. The molecular mechanisms are not clear, even if there is increasing evidence for an important action of nitric oxide (NO), probably mediated by the regulation of the gene encoding for vascular endothelial growth factor (VEGF). The aim of our study was to investigate the immunohistochemical expression of VEGF and nitric oxide synthase (NOS) isoforms in human skin-derived MSCs, as well as the production of VEGF and NO, because these cells are less well characterized than bone marrow MSCs. MSCs were obtained from skin biopsies of healthy adult patients undergoing cosmetic plastic surgery, expanded and characterized for specific surface antigens. The cells were then evaluated for the immunohistochemical expression of VEGF, and NOS isoforms, as well as for VEGF and NO secretion in cell culture medium. Our immunohistochemical analysis showed that proliferating MSCs derived from human skin exhibit VEGF expression at cytoplasmic level as well as cytosolic and nuclear localization of all the three isoforms of NOS, even if with different patterns. In addition, our data evidenced the release of both VEGF and NO in cell culture supernatants. In conclusion, our results suggest that a therapeutic approach based on the human skin-derived MSCs may have a positive effect in wound healing conditions, through their ability to provide VEGF and NO to the damaged area.

Functional Characterization of Calcium-Signaling Pathways of Human Skin-Derived Mesenchymal Stem Cells

Background: Mesenchymal stem cells (MSCs) derived from adult human tissues are able to differentiate into various specialized cell types. In research, they can therefore be used like embryonic cells but without the ethical restrictions. Among the various human tissues, skin as a source is characterized by great accessibility and availability using noninvasive procedures and is without the risk of oncogenesis after transplantation. The recent isolation of MSCs has shown the lack of knowledge regarding their specific features, including the calcium-signaling pathways. Methods: In this study, we isolated MSCs from human skin biopsies (S-MSCs) and characterized them phenotypically and their calcium-signaling pathways by the means of Ca²⁺ imaging and video microscopic experiments. Results: The cytofluorimetric analysis of the expression of surface markers on S-MSCs revealed that they express the normal pattern present on MSCs. Interestingly, these cells appeared to be successfully cryopreserved at early passages. Calcium imaging on single S-MSCs shows that these cells did not display significant spontaneous activity or a response to a depolarizing agent. However, ATP or acetylcholine-induced intracellular calcium increase via ionotropic or metabotropic receptors, respectively. Conclusion: The results presented here reveal that S-MSCs show morphological and functional features that make them useful as an in vitro model to study cell differentiation.

Characterization of human skin-derived mesenchymal stem cell proliferation rate in different growth conditions.

This study investigated conditions for optimal in vitro propagation of human skin-derived mesenchymal stem cells (S-MSC). Forty primary skin-derived precursor cell (SKP) cultures were established from both male and female donors (age 29-65 years) and eight of them were randomly selected for in-depth characterization. Effects of basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), leukemia inhibiting factor (LIF) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) on S-MSC proliferation were investigated. Primary SKP cultures were >95% homogenous for CD90, CD73, and CD105 marker expression enabling to classify these cells as S-MSC. FGF-2 dose-dependent stimulation was observed in low serum medium only, whereas EGF neither stimulated S-MSC proliferation nor potentates the effect of FGF-2. Pronounced donor to donor differences among S-MSC cultures were observed in 3-day proliferation assay. This study demonstrates that homogenous S-MSC populations can be reproducibly isolated from individual donors of different age. Optimal cell culture conditions for in vitro propagation of S-MSC are B27 supplemented or low serum media with FGF-2 (4 ng/ml). EGF and LIF as well as db-cAMP are dispensable for S-MSC proliferation.