J Korean Assoc Oral Maxillofac Surg.  2012 Dec;38(6):343-353. 10.5125/jkaoms.2012.38.6.343.

Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro

• Byun JH
• Kang EJ
• Park SC
• Kang DH
• Choi MJ
• Rho GJ
• Park BW

• KMID: 2136971
• DOI: http://doi.org/10.5125/jkaoms.2012.38.6.343

Abstract

OBJECTIVES
This aim of this study was to effectively isolate mesenchymal stem cells (hSMSCs) from human submandibular skin tissues (termed hSMSCs) and evaluate their characteristics. These hSMSCs were then chemically induced to the neuronal lineage and analyzed for their neurogenic characteristics in vitro.
MATERIALS AND METHODS
Submandibular skin tissues were harvested from four adult patients and cultured in stem cell media. Isolated hSMSCs were evaluated for their multipotency and other stem cell characteristics. These cells were differentiated into neuronal cells with a chemical induction protocol. During the neuronal induction of hSMSCs, morphological changes and the expression of neuron-specific proteins (by fluorescence-activated cell sorting [FACS]) were evaluated.
RESULTS
The hSMSCs showed plate-adherence, fibroblast-like growth, expression of the stem-cell transcription factors Oct 4 and Nanog, and positive staining for mesenchymal stem cell (MSC) marker proteins (CD29, CD44, CD90, CD105, and vimentin) and a neural precursor marker (nestin). Moreover, the hSMSCs in this study were successfully differentiated into multiple mesenchymal lineages, including osteocytes, adipocytes, and chondrocytes. Neuron-like cell morphology and various neural markers were highly visible six hours after the neuronal induction of hSMSCs, but their neuron-like characteristics disappeared over time (24-48 hrs). Interestingly, when the chemical induction medium was changed to Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS), the differentiated cells returned to their hSMSC morphology, and their cell number increased. These results indicate that chemically induced neuron-like cells should not be considered true nerve cells.
CONCLUSION
Isolated hSMSCs have MSC characteristics and express a neural precursor marker, suggesting that human skin is a source of stem cells. However, the in vitro chemical neuronal induction of hSMSC does not produce long-lasting nerve cells and more studies are required before their use in nerve-tissue transplants.

Keyword

Skin; Mesenchymal stem cell; In vitro neuronal differentiation

MeSH Terms

Adipocytes
Adult
Cell Count
Chondrocytes
Eagles
Flow Cytometry
Humans
Mesenchymal Stromal Cells
Neurons
Osteocytes
Proteins
Skin
Stem Cells
Transcription Factors
Transplants
Proteins
Transcription Factors

Figure

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  Fig. 1 Isolation and primary culture of human skin-derived cells with serum-containing adherent cell culture method (scale bar=100 µm). A. Harvested submandibular skin tissue. B-D. Culturing human skin-derived cells (hSDCs) on the 3rd (B), 7th (C), and 14th (D) day can be observed during primary culture (P0). B. Irregular and heterogeneous hSDCs isolated from a skin fragment (black shadow) in the primary culture plates. C. After 7 days of P0, proliferating irregularly shaped hSDCs were detected in the plates. D. After about 2 weeks of P0, plate-adherent, fibroblast-like homogeneous cells were detected in the culture plates.

• 

  Fig. 2 Expression of early transcription factors Oct 4, Nanog, and Sox 2 by immunocytochemistry (A: scale bar=100 µm) and reverse transcription-polymerase chain reaction (B) in human skin-derived cells (hSDCs) at passage 3. Positive expression of Oct 4 and Nanog, even though Sox 2 was hardly expressed, indicates that the hSDCs in this study are multipotential primitive cells. (GAPDH: glyceraldehyde 3-phosphate dehydrogenase)

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  Fig. 3 Fluorescence-activated cell sorting analysis of cultured human skin-derived cells. Skin-derived cells at passage 3 were positive for specific mesenchymal stem cell markers (CD29, CD44, CD90, CD105, and vimentin) and neural precursor cell marker (nestin). Open histograms represent staining with negative control, with the black histograms depicting the fluorescence intensity of each of the cell surface antibodies.

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  Fig. 4 Mesenchymal-lineage differentiations of human skin-derived mesenchymal stem cells (hSMSCs) into ostocytes (a, b), adiopcytes (c), and chondrocytes (d) for 4 weeks (A: scale bar=100 µm). A. In vitro differentiated cells showed positive staining in the specific staining methods. (a, b) Calcium deposits were observed on the cell surface by von Kossa (a) and Alizalin red (b) staining. (c) Lipid droplets were noted in the cytoplasm of cells by Oil red O staining. (d) Proteoglycans were confirmed on the cell surface using Alcian blue. B. Reverse transcription-polymerase chain reaction results for in vitro differentiated osteocytes and adipocytes from hSMSCs. (a) ON and OC were detected in osteogenic differentiated cells. (b) PPARγ2 and aP2 were expressed in adipogenic differentiated cells. (OC: osteocalcin, ON: osteonectin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, aP2: adipocyte protein 2)

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  Fig. 5 The upper graph illustrates the schematic in vitro neural induction protocol used in this study. Cultured hSMSCs at passage 3 were preinduced for 24 hrs. The experimental group was neurally induced by a chemical protocol for 48 hr. In the control group, 24 hr after neural induction, the inductive medium was changed to DMEM supplemented with 20% FBS, and morphologic changes were then observed after an additional 24 hrs of media change. A-E. The microphotographs show the morphologic changes of hSMSCs after chemical neural induction (scale bar=100 µm). A. Immediately after neuronal preinduction (0 hr). There are no remarkable morphological changes compared to the original hSMSCs. B. Six hours (6 hr) after neural induction, the neuron-like cells exhibit peak activity. C, D. After the passage of neural induction time (24 and 48 hrs post-neural induction), the neuron-like cells decreased in number, and their shape deteriorated. E. In the control cells, neural differentiated cells returned to the original hSMSC morphology, and cell number increased 24 hrs after media change as DMEM with 20% FBS. F. The number of cells decreased with the passage of neural induction time, but the number increased after the inductive medium was changed. (hSMSCs: human skin-derived mesenchymal stem cells, bFGF: basic fibroblast growth factor, FBS: fetal bovine serum, DMEM: Dulbecco's Modified Eagle Medium, DMSO: dimethylsulfoxide, VPA: valporic acid, BHA: butylated hydroxyanisole)

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  Fig. 6 A. Immunocytochemical studies for various neuronal and angiogenic marker proteins after the in vitro chemical neural induction of hSMSCs (scale bar=100 µm). Most marker proteins were highly visible 6 hrs after neural induction. Nestin was expressed in the 0 hr specimen (before nerve induction), which is similar to the result of FACS analysis. NGF and VEGF were highly visible with their receptors (p75NGFR, trkA, VEGFR1, and VEGFR2) during neuronal differentiation. B. Immunocytochemical intensities for specific proteins. The expression of most proteins, except NeuN, peaked 6 hrs after neural induction, and then decreased over time (24 hrs and 48 hrs after induction). Data represent the mean±SE of four independent experiments. A star (*) indicates a significant difference from the control (P<0.05). (S-100: S-100 protein, NF: neurofilament, MBP: myelin basic protein, NeuN: neural-specific nuclear protein, NGF: nerve growth factor, p75NGFR: p75 nerve growth factor receptor, trkA: tyrosine kinase receptor A, VEGF: vascular endothelial cell growth factor, VEGFR: vascular endothelial cell growth factor receptor)

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