Isolation and Characterization of Neurogenic Mesenchymal Stem Cells in Human Scalp Tissue
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《干细胞学杂志》
a Center for Stem Cell Research, Wan-Fang Hospital, Taipei, Taiwan;
b Graduate Institute of Cell and Molecular Biology, Taipei, Taiwan;
c Wan-Fang Hospital, Taipei, Taiwan;
d School of Pharmaceutical Sciences, Taipei Medical University, Taipei, Taiwan
Key Words. Scalp tissue ? Multipotent stem/progenitor cells ? Neurogenic differentiation
Correspondence: Daniel Tzu-bi Shih, Ph.D., Graduate Institute of Cell and Molecular Biology, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan 110. Telephone: 886-2-2377-8619; Fax: 886-2-2377-8620; e-mail: cmbdshih@tmu.edu.tw
ABSTRACT
Stem cells isolated from developing tissues can be differentiated into more than one specific cell type . The proportion of these multipotent stem cells diminishes with the maturation of the tissue. On the other hand, recent studies have shown that adult tissues contain residual tissue stem/progenitor cells capable of not only generating mature cells of their own tissue but also renewing other tissue cells. A growing body of evidence has also shown that the process of tissue repair is driven by these stem-like cells residing in different tissues. Among candidates for reparative cells are the stem cells from adult bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (BM-MSCs). BM-MSCs are defined by their ability to differentiate into cells of osteogenic, chondrogenic, adipogenic, and, more or less, myogenic lineages. Besides bone marrow , the peripheral blood , the retina , the adipose tissue , and the central nervous system are known reservoirs for multipotent, mesenchymal stem cells (MSCs). The adherent, spreading morphology of MSCs is visibly distinct from hematopoietic stem cells. Our current interest is focused on identifying new sources of MSCs and characterizing their potentials for differentiating into nonmesenchymal tissues.
Recently, BM-MSCs were found to undergo neuronal differentiation when they were cocultured with brain tissue in the absence of hematopoietic stem cells . The study implied that a population of cells with neuroectodermal potential can be derived from marrow stroma, the mesodermal mesenchyme origin. Successively, several groups have reported that circulating blood is a reservoir of multipotent MSCs that can be directed into adipogenic, osteogenic, myogenic, neurogenic, hepatogenic or epithelial, endothelial, hepatogenic lineages .
In vitro characterization and maintenance of tissue stem/ progenitor cells is critical to the assessment of their potential for clinical applications. Murine skin-derived progenitors (mSKPs) obtained during developmental stages showed osteogenic, adipogenic, smooth muscle, and neuronal differentiation potentials , suggesting that they could be exploited as an alternative source for treating mesenchymal and neurodegenerative disorders. Here we describe that the human scalp tissue contains stem/progenitor cells with mesenchymal and neurogenic differentiation potentials. We isolated the scalp-derived adherent cells (hSCPs) by both medium-selective (ms-hSCPs) and clongenic (c-hSCPs) cultures, characterized their growth kinetics, mesenchymal differentiation potentials, and expression of cell markers, and concluded their characteristics as neurogenic mesenchymal stem/progenitor cells.
MATERIALS AND METHODS
Cell Isolation and Characterization
The total amount of cells isolated from each scalp tissue sample ranged from 5 x 104 to 1 x 105 cells. Approximately 0.5%–2% of the isolated tissue cells was found to be the adherent hSCPs. Morphologically, the ms-hSCP or c-hSCP cells were less flattened and fibroblast-like compared with BM-MSCs (Figs. 1A, 1B). Growth rate for c-hSCPs and BM-MSCs was comparable, as shown in Fig. 1C. The doubling time of ms-hSCPs and c-hSCPs derived from human scalp was approximately 40 hours, whereas that of BM-MSCs was approximately 48 hours. Similarities of hSCP morphology and growth kinetics were observed in more than 30 tissue isolations (one patient per isolation). No significant difference of growth rate, cell frequency, and doubling time was observed among these scalp tissue isolations (data not shown).
Figure 1. Morphology and cell growth rate of scalp-derived clongenic stem cells (c-hSCPs) (A) and bone marrow–derived mesenchymal stem cells (BM-MSCs) (B). Representative micrographs (A, B) and growth rates of c-hSCPs () and BM-MSCs () are illustrated (C). After day 35, both c-hSCPs and BM-MSCs continuously proliferated until the cell size and growth rate became large and slow.
Both c-hSCPs and ms-hSCPs expressed an immune-phenotype antigen profile similar to BM-MSCs and shared common cell markers, as shown in Table 1 and Figure 2, including the following: MSC: CDw90, SH2, and SH4 but not Stro-1; hematopoietic: CD45–, CD38–, and CD34–; HLA: class I+; hyaluronate receptor: CD44+; integrins: CD49d+, CD49e+, CD49f+; ALCAM: CD166+; angiogenic: CD105+, CD31–; and cytokine receptors: EGFR+, PDGFR+. Notably, a slightly higher frequency of cells of hSCPs expressed PDGFR and CD49f antigens compared with the BM-MSCs.
Figure 2. Fluorescence-activated cell sorting analysis of scalp-derived clongenic stem cell surface markers.
Mesenchymal Differentiation of c-hSCPs and ms-hSCPs
We observed that c-hSCPs and ms-hSCPs exhibit similar mesenchymal differentiation capacity as BM-MSCs do.
Osteogenesis ? To examine whether hSCPs were as capable of mesenchymal differentiation as the BM-MSCs, c-hSCPs and ms-hSCPs were subjected to osteogenic lineage differentiation. After 1–2 weeks of differentiation in culture, osteogenic-like cell structure gradually formed and stained positive for von Kossa staining. The calcium phosphate formed in these cells precipitated along the cell membrane and showed up as brown, large, aggregate particles embedded in the extracellular matrix when stained with von Kossa (Fig. 3).
Figure 3. Osteogenic differentiation of single-cell scalp-derived clongenic stem cells (c-hSCPs), medium-selection scalp-derived stem cells (ms-hSCPs), and bone marrow mesenchymal stem cells (BM-MSCs). After 14 days, a calcified extracellular matrix (arrow, dark black area) was present and positive for von Kossa staining.
In the calcium incorporation assay, the amount of calcium phosphate precipitation increased from 2.38 μg per 105 cells to 20 μg per 105 cells in c-hSCP osteogenesis, whereas in BM-MSCs osteogenesis, it increased from 0.01 μg per 105 cells to 7 μg per 105 cells. Alkaline phosphate staining produced similar results (data not shown). We therefore concluded that c-hSCPs were capable of similar osteogenic differentiation as the BM-MSCs.
Chondrogenesis ? Chondrogenic differentiation was achieved by dropping high-density (5 x 105 to 1 x 106) c-hSCPs and ms-hSCPs or BM-MSCs on the dish center containing chondrogenic differentiation medium. The cell condensed and formed chondrosphere-like pellets in 3 days. Frozen dissected specimens were collected after 2 weeks in differentiation culture. Alcian Blue staining was used to visualize mucopolyglycan formation in histological dissections of the above cultures. Mucopolyglycan was present in cartilaginous matrices and lacunae with extracellular proteoglycan formation (Fig. 4). In addition, positive Safranin-O staining was observed for condensed sulfate proteoglycans formation in both BM-MSC and hSCP cultures (Fig. 4).
Figure 4. Chondrosphere formation in single-cell scalp-derived clongenic stem cells (c-hSCPs), medium-selection scalp-derived stem cells (ms-hSCPs), and bone marrow mesenchymal stem cells (BM-MSCs). Typical chondrosphere formation was observed for c-hSCPs, ms-hSCPs, and BM-MSCs. The acidic mucopolysaccharide was stained with Alcian blue, whereas proteoglycans were stained with Safranin-O. c-hSCPs, ms-hSCPs, and BM-MSCs were positive for both assays.
Adipogenesis ? Small oil droplets were observed to appear gradually in the cytoplasm after 1 week of adipogenic induction in both BM-MSC and hSCP cultures. Adipocyte characteristics were confirmed by positive staining with Oil Red O after 2 weeks of induction but to a lesser extent in hSCPs compared with BM-MSCs (Fig. 5).
Figure 5. Adipogenic differentiation of single-cell scalp-derived clongenic stem cells (c-hSCPs), medium-selection scalp-derived stem cells (ms-hSCPs), and bone marrow mesenchymal stem cells (BM-MSCs). Cells with the same passage numbers, as in previous assays, were used. Intracellular oil droplets formed and were stained red by Oil Red O for c-hSCPs, ms-hSCPs, and BM-MSCs (arrow, red area).
Neuronal Differentiation Potential of hSCPs
Both hSCPs and BM-MSCs exhibit more or less capability for differentiation along all three mesenchymal lineages. We wondered whether hSCPs display a neurogenic differentiation potential like the reported rodent SKPs . As predicted, hSCPs exhibited significant neurogenic differentiation potential, because more than 80% of the viable c-hSCP displayed morphological changes into contracted cell bodies and elaborate processes (Figs. 6A, 6B) when cultured in a neurogenic induction media. Immunofluorescence staining of NSE provided additional evidence for neuron-differentiating characteristics (Fig. 6C). RT-PCR analysis of neurogenic and early gene expression in c-hSCP culture showed upregulation of MAP2 and NF-M and downregulation of GFAP and early progenitor hSOX2 gene (Fig. 7). These results demonstrated that c-hSCPs are indeed capable of differentiation into neurogenic precursors more efficiently than the BM-MSCs are.
Figure 6. Neurogenic differentiation of single-cell scalp-derived clongenic stem cells (c-hSCPs) on poly-L-lysine–coated dishes. Cell morphology before differentiation (A) and after 48 hours of induction in NC210 neurogenic culture media (B). Neurogenic neuron-specific enolase (NSE) expression was confirmed by fluoro-NSE monoclonal antibody (mAb) staining (C). Top panels show c-hSCPs (P4) differentiated into NSE+ neurosphere-like cells (>80%) at various parts of the culture dish after 5 days of induction in NC210 culture condition. Bottom panels show the same cells counterstained by nucleic DAPI as contrasts. Neurogenic differentiation of bone marrow mesenchymal stem cells (30%) confirmed by fluoro-NSE mAb staining as a comparable study (D).
Figure 7. Neurogenic-specific genes microtubule-associated protein 2 (MAP2), NCAM, neuron filament-M (NF-M), glial fibrillary acid protein (GFAP), and the early progenitor hSOX2 gene were examined by reverse transcription–polymerase chain reaction analyses. Upregulation of neuronal-specific MAP2 and NF-M and downregulation of glial GFAP and early progenitor hSOX2 gene expressions were detected after neuronal-induction treatment of the single-cell scalp-derived clongenic stem cells (c-hSCPs) in NC210 medium. Target genes were visible at 18 cycles (beta actin), 25 cycles (NCAM, MAP2, and NF-M), and 30 cycles (GFAP and hSOX2), respectively.
Interestingly, GFAP transcription can be detected in the untreated hSCPs but disappeared after subsequent neurongenic differentiation under NC210 culture treatment. Untreated BM-MSCs failed to show detectable GFAP transcripts, although to a lesser extent they can also be differentiated into neuron precursors. We suspected that c-hSCPs behave as a multipotent neuron progenitor cell that could differentiate into both neuron and glial and mesenchymal lineages depending on their chemical treatment, because GFAP transcripts of hSCPs can be detected under different culture conditions (data not shown).
DISCUSSION
Fuchs E, Segre JA. Stem cells: a new lease on life. Cell 2000;100:143–155.
Wexler SA, Donaldson C, Denning-Kendall P et al. Adult bone marrow is a rich source of human mesenchymal "stem" cells but umbilical cord and mobilized adult blood are not. Br J Haematol 2003;121:368–374.
Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999;181:67–73.
Tropepe V, Coles BL, Chiasson BJ et al. Retinal stem cells in the adult mammalian eye. Science 2000;287:2032–2036.
Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multi-potent stem cells. Mol Biol Cell 2002;13:4279–4295.
Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–228.
Ashjian PH, Elbarbary AS, Edmonds B et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg 2003;111:1922–1931.
Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res 2002;69:908–917.
Gage FH. Mammalian neural stem cells. Science 2000;287:1433–1438.
Cardozo-Pelaez F, Song S, Parthasarathy A et al. Attenuation of age-dependent oxidative damage to DNA and protein in brainstem of Tg Cu/Zn SOD mice. Neurobiol Aging 1998;19:311–316.
Goodwin HS, Bicknese AR, Chien SN et al. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 2001;7:581–588.
De Ugarte DA, Morizono K, Elbarbary A et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 2003;174:101–109.
Mizuno H, Zuk PA, Zhu M et al. Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg 2002;109:199–209.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Suva D, Garavaglia G, Menetrey J et al. Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells. J Cell Physiol 2004;198:110–118.
Toma JG, Akhavan M, Fernandes KJ et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001;3:778–784.
Jahoda CA, Whitehouse J, Reynolds AJ et al. Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages. Exp Dermatol 2003;12:849–859.
Banfi A, Muraglia A, Dozin B et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 2000;28:707–715.
Dai C, Celestino JC, Okada Y et al. PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev 2001;15:1913–1925.
Cai J, Wu Y, Mirua T et al. Properties of a fetal multipotent neural stem cell (NEP cell). Dev Biol 2002;251:221–240.
Hall A, Giese NA, Richardson WD. Spinal cord oligodendrocytes develop from ventrally derived progenitor cells that express PDGF alpha-receptors. Development 1996;122:4085–4094.
Gary DS, Mattson MP. Integrin signaling via the PI3-kinase-Akt pathway increases neuronal resistance to glutamate-induced apoptosis. J Neurochem 2001;76:1485–1496.
Frost EE, Buttery PC, Milner R et al. Integrins mediate a neuronal survival signal for oligodendrocytes. Curr Biol 1999;9:1251–1254.
Calza L, Giuliani A, Fernandez M et al. Neural stem cells and cholinergic neurons: regulation by immunolesion and treatment with mitogens, retinoic acid, and nerve growth factor. Proc Natl Acad Sci U S A 2003;100:7325–7330.
Kim BJ, Seo JH, Bubien JK et al. Differentiation of adult bone marrow stem cells into neuroprogenitor cells in vitro. Neuroreport 2002;13:1185–1188.
Duittoz AH, Hevor T. Primary culture of neural precursors from the ovine central nervous system (CNS). J Neurosci Methods 2001;107:131–140.
Brunner G, Gabrilove J, Rifkin DB et al. Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan. J Cell Biol 1991;114:1275–1283.
Brunner G, Nguyen H, Gabrilove J et al. Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells. Blood 1993;81:631–638.
Oliver JA. Adenylate cyclase and protein kinase C mediate opposite actions on endothelial junctions. J Cell Physiol 1990;145:536–542.
Ding I, Huang K, Wang X et al. Radioprotection of hematopoietic tissue by fibroblast growth factors in fractionated radiation experiments. Acta Oncol 1997;36:337–340.
Zhang H, Wang JZ, Sun HY et al. The effects of GM1 and bFGF synergistically inducing adult rat bone marrow stromal cells to form neural progenitor cells and their differentiation. Chin J Traumatol 2004;7:3–6.(Daniel Tzu-bi Shiha,b, Do)
b Graduate Institute of Cell and Molecular Biology, Taipei, Taiwan;
c Wan-Fang Hospital, Taipei, Taiwan;
d School of Pharmaceutical Sciences, Taipei Medical University, Taipei, Taiwan
Key Words. Scalp tissue ? Multipotent stem/progenitor cells ? Neurogenic differentiation
Correspondence: Daniel Tzu-bi Shih, Ph.D., Graduate Institute of Cell and Molecular Biology, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan 110. Telephone: 886-2-2377-8619; Fax: 886-2-2377-8620; e-mail: cmbdshih@tmu.edu.tw
ABSTRACT
Stem cells isolated from developing tissues can be differentiated into more than one specific cell type . The proportion of these multipotent stem cells diminishes with the maturation of the tissue. On the other hand, recent studies have shown that adult tissues contain residual tissue stem/progenitor cells capable of not only generating mature cells of their own tissue but also renewing other tissue cells. A growing body of evidence has also shown that the process of tissue repair is driven by these stem-like cells residing in different tissues. Among candidates for reparative cells are the stem cells from adult bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (BM-MSCs). BM-MSCs are defined by their ability to differentiate into cells of osteogenic, chondrogenic, adipogenic, and, more or less, myogenic lineages. Besides bone marrow , the peripheral blood , the retina , the adipose tissue , and the central nervous system are known reservoirs for multipotent, mesenchymal stem cells (MSCs). The adherent, spreading morphology of MSCs is visibly distinct from hematopoietic stem cells. Our current interest is focused on identifying new sources of MSCs and characterizing their potentials for differentiating into nonmesenchymal tissues.
Recently, BM-MSCs were found to undergo neuronal differentiation when they were cocultured with brain tissue in the absence of hematopoietic stem cells . The study implied that a population of cells with neuroectodermal potential can be derived from marrow stroma, the mesodermal mesenchyme origin. Successively, several groups have reported that circulating blood is a reservoir of multipotent MSCs that can be directed into adipogenic, osteogenic, myogenic, neurogenic, hepatogenic or epithelial, endothelial, hepatogenic lineages .
In vitro characterization and maintenance of tissue stem/ progenitor cells is critical to the assessment of their potential for clinical applications. Murine skin-derived progenitors (mSKPs) obtained during developmental stages showed osteogenic, adipogenic, smooth muscle, and neuronal differentiation potentials , suggesting that they could be exploited as an alternative source for treating mesenchymal and neurodegenerative disorders. Here we describe that the human scalp tissue contains stem/progenitor cells with mesenchymal and neurogenic differentiation potentials. We isolated the scalp-derived adherent cells (hSCPs) by both medium-selective (ms-hSCPs) and clongenic (c-hSCPs) cultures, characterized their growth kinetics, mesenchymal differentiation potentials, and expression of cell markers, and concluded their characteristics as neurogenic mesenchymal stem/progenitor cells.
MATERIALS AND METHODS
Cell Isolation and Characterization
The total amount of cells isolated from each scalp tissue sample ranged from 5 x 104 to 1 x 105 cells. Approximately 0.5%–2% of the isolated tissue cells was found to be the adherent hSCPs. Morphologically, the ms-hSCP or c-hSCP cells were less flattened and fibroblast-like compared with BM-MSCs (Figs. 1A, 1B). Growth rate for c-hSCPs and BM-MSCs was comparable, as shown in Fig. 1C. The doubling time of ms-hSCPs and c-hSCPs derived from human scalp was approximately 40 hours, whereas that of BM-MSCs was approximately 48 hours. Similarities of hSCP morphology and growth kinetics were observed in more than 30 tissue isolations (one patient per isolation). No significant difference of growth rate, cell frequency, and doubling time was observed among these scalp tissue isolations (data not shown).
Figure 1. Morphology and cell growth rate of scalp-derived clongenic stem cells (c-hSCPs) (A) and bone marrow–derived mesenchymal stem cells (BM-MSCs) (B). Representative micrographs (A, B) and growth rates of c-hSCPs () and BM-MSCs () are illustrated (C). After day 35, both c-hSCPs and BM-MSCs continuously proliferated until the cell size and growth rate became large and slow.
Both c-hSCPs and ms-hSCPs expressed an immune-phenotype antigen profile similar to BM-MSCs and shared common cell markers, as shown in Table 1 and Figure 2, including the following: MSC: CDw90, SH2, and SH4 but not Stro-1; hematopoietic: CD45–, CD38–, and CD34–; HLA: class I+; hyaluronate receptor: CD44+; integrins: CD49d+, CD49e+, CD49f+; ALCAM: CD166+; angiogenic: CD105+, CD31–; and cytokine receptors: EGFR+, PDGFR+. Notably, a slightly higher frequency of cells of hSCPs expressed PDGFR and CD49f antigens compared with the BM-MSCs.
Figure 2. Fluorescence-activated cell sorting analysis of scalp-derived clongenic stem cell surface markers.
Mesenchymal Differentiation of c-hSCPs and ms-hSCPs
We observed that c-hSCPs and ms-hSCPs exhibit similar mesenchymal differentiation capacity as BM-MSCs do.
Osteogenesis ? To examine whether hSCPs were as capable of mesenchymal differentiation as the BM-MSCs, c-hSCPs and ms-hSCPs were subjected to osteogenic lineage differentiation. After 1–2 weeks of differentiation in culture, osteogenic-like cell structure gradually formed and stained positive for von Kossa staining. The calcium phosphate formed in these cells precipitated along the cell membrane and showed up as brown, large, aggregate particles embedded in the extracellular matrix when stained with von Kossa (Fig. 3).
Figure 3. Osteogenic differentiation of single-cell scalp-derived clongenic stem cells (c-hSCPs), medium-selection scalp-derived stem cells (ms-hSCPs), and bone marrow mesenchymal stem cells (BM-MSCs). After 14 days, a calcified extracellular matrix (arrow, dark black area) was present and positive for von Kossa staining.
In the calcium incorporation assay, the amount of calcium phosphate precipitation increased from 2.38 μg per 105 cells to 20 μg per 105 cells in c-hSCP osteogenesis, whereas in BM-MSCs osteogenesis, it increased from 0.01 μg per 105 cells to 7 μg per 105 cells. Alkaline phosphate staining produced similar results (data not shown). We therefore concluded that c-hSCPs were capable of similar osteogenic differentiation as the BM-MSCs.
Chondrogenesis ? Chondrogenic differentiation was achieved by dropping high-density (5 x 105 to 1 x 106) c-hSCPs and ms-hSCPs or BM-MSCs on the dish center containing chondrogenic differentiation medium. The cell condensed and formed chondrosphere-like pellets in 3 days. Frozen dissected specimens were collected after 2 weeks in differentiation culture. Alcian Blue staining was used to visualize mucopolyglycan formation in histological dissections of the above cultures. Mucopolyglycan was present in cartilaginous matrices and lacunae with extracellular proteoglycan formation (Fig. 4). In addition, positive Safranin-O staining was observed for condensed sulfate proteoglycans formation in both BM-MSC and hSCP cultures (Fig. 4).
Figure 4. Chondrosphere formation in single-cell scalp-derived clongenic stem cells (c-hSCPs), medium-selection scalp-derived stem cells (ms-hSCPs), and bone marrow mesenchymal stem cells (BM-MSCs). Typical chondrosphere formation was observed for c-hSCPs, ms-hSCPs, and BM-MSCs. The acidic mucopolysaccharide was stained with Alcian blue, whereas proteoglycans were stained with Safranin-O. c-hSCPs, ms-hSCPs, and BM-MSCs were positive for both assays.
Adipogenesis ? Small oil droplets were observed to appear gradually in the cytoplasm after 1 week of adipogenic induction in both BM-MSC and hSCP cultures. Adipocyte characteristics were confirmed by positive staining with Oil Red O after 2 weeks of induction but to a lesser extent in hSCPs compared with BM-MSCs (Fig. 5).
Figure 5. Adipogenic differentiation of single-cell scalp-derived clongenic stem cells (c-hSCPs), medium-selection scalp-derived stem cells (ms-hSCPs), and bone marrow mesenchymal stem cells (BM-MSCs). Cells with the same passage numbers, as in previous assays, were used. Intracellular oil droplets formed and were stained red by Oil Red O for c-hSCPs, ms-hSCPs, and BM-MSCs (arrow, red area).
Neuronal Differentiation Potential of hSCPs
Both hSCPs and BM-MSCs exhibit more or less capability for differentiation along all three mesenchymal lineages. We wondered whether hSCPs display a neurogenic differentiation potential like the reported rodent SKPs . As predicted, hSCPs exhibited significant neurogenic differentiation potential, because more than 80% of the viable c-hSCP displayed morphological changes into contracted cell bodies and elaborate processes (Figs. 6A, 6B) when cultured in a neurogenic induction media. Immunofluorescence staining of NSE provided additional evidence for neuron-differentiating characteristics (Fig. 6C). RT-PCR analysis of neurogenic and early gene expression in c-hSCP culture showed upregulation of MAP2 and NF-M and downregulation of GFAP and early progenitor hSOX2 gene (Fig. 7). These results demonstrated that c-hSCPs are indeed capable of differentiation into neurogenic precursors more efficiently than the BM-MSCs are.
Figure 6. Neurogenic differentiation of single-cell scalp-derived clongenic stem cells (c-hSCPs) on poly-L-lysine–coated dishes. Cell morphology before differentiation (A) and after 48 hours of induction in NC210 neurogenic culture media (B). Neurogenic neuron-specific enolase (NSE) expression was confirmed by fluoro-NSE monoclonal antibody (mAb) staining (C). Top panels show c-hSCPs (P4) differentiated into NSE+ neurosphere-like cells (>80%) at various parts of the culture dish after 5 days of induction in NC210 culture condition. Bottom panels show the same cells counterstained by nucleic DAPI as contrasts. Neurogenic differentiation of bone marrow mesenchymal stem cells (30%) confirmed by fluoro-NSE mAb staining as a comparable study (D).
Figure 7. Neurogenic-specific genes microtubule-associated protein 2 (MAP2), NCAM, neuron filament-M (NF-M), glial fibrillary acid protein (GFAP), and the early progenitor hSOX2 gene were examined by reverse transcription–polymerase chain reaction analyses. Upregulation of neuronal-specific MAP2 and NF-M and downregulation of glial GFAP and early progenitor hSOX2 gene expressions were detected after neuronal-induction treatment of the single-cell scalp-derived clongenic stem cells (c-hSCPs) in NC210 medium. Target genes were visible at 18 cycles (beta actin), 25 cycles (NCAM, MAP2, and NF-M), and 30 cycles (GFAP and hSOX2), respectively.
Interestingly, GFAP transcription can be detected in the untreated hSCPs but disappeared after subsequent neurongenic differentiation under NC210 culture treatment. Untreated BM-MSCs failed to show detectable GFAP transcripts, although to a lesser extent they can also be differentiated into neuron precursors. We suspected that c-hSCPs behave as a multipotent neuron progenitor cell that could differentiate into both neuron and glial and mesenchymal lineages depending on their chemical treatment, because GFAP transcripts of hSCPs can be detected under different culture conditions (data not shown).
DISCUSSION
Fuchs E, Segre JA. Stem cells: a new lease on life. Cell 2000;100:143–155.
Wexler SA, Donaldson C, Denning-Kendall P et al. Adult bone marrow is a rich source of human mesenchymal "stem" cells but umbilical cord and mobilized adult blood are not. Br J Haematol 2003;121:368–374.
Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999;181:67–73.
Tropepe V, Coles BL, Chiasson BJ et al. Retinal stem cells in the adult mammalian eye. Science 2000;287:2032–2036.
Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multi-potent stem cells. Mol Biol Cell 2002;13:4279–4295.
Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–228.
Ashjian PH, Elbarbary AS, Edmonds B et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg 2003;111:1922–1931.
Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res 2002;69:908–917.
Gage FH. Mammalian neural stem cells. Science 2000;287:1433–1438.
Cardozo-Pelaez F, Song S, Parthasarathy A et al. Attenuation of age-dependent oxidative damage to DNA and protein in brainstem of Tg Cu/Zn SOD mice. Neurobiol Aging 1998;19:311–316.
Goodwin HS, Bicknese AR, Chien SN et al. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 2001;7:581–588.
De Ugarte DA, Morizono K, Elbarbary A et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 2003;174:101–109.
Mizuno H, Zuk PA, Zhu M et al. Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg 2002;109:199–209.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Suva D, Garavaglia G, Menetrey J et al. Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells. J Cell Physiol 2004;198:110–118.
Toma JG, Akhavan M, Fernandes KJ et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001;3:778–784.
Jahoda CA, Whitehouse J, Reynolds AJ et al. Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages. Exp Dermatol 2003;12:849–859.
Banfi A, Muraglia A, Dozin B et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 2000;28:707–715.
Dai C, Celestino JC, Okada Y et al. PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev 2001;15:1913–1925.
Cai J, Wu Y, Mirua T et al. Properties of a fetal multipotent neural stem cell (NEP cell). Dev Biol 2002;251:221–240.
Hall A, Giese NA, Richardson WD. Spinal cord oligodendrocytes develop from ventrally derived progenitor cells that express PDGF alpha-receptors. Development 1996;122:4085–4094.
Gary DS, Mattson MP. Integrin signaling via the PI3-kinase-Akt pathway increases neuronal resistance to glutamate-induced apoptosis. J Neurochem 2001;76:1485–1496.
Frost EE, Buttery PC, Milner R et al. Integrins mediate a neuronal survival signal for oligodendrocytes. Curr Biol 1999;9:1251–1254.
Calza L, Giuliani A, Fernandez M et al. Neural stem cells and cholinergic neurons: regulation by immunolesion and treatment with mitogens, retinoic acid, and nerve growth factor. Proc Natl Acad Sci U S A 2003;100:7325–7330.
Kim BJ, Seo JH, Bubien JK et al. Differentiation of adult bone marrow stem cells into neuroprogenitor cells in vitro. Neuroreport 2002;13:1185–1188.
Duittoz AH, Hevor T. Primary culture of neural precursors from the ovine central nervous system (CNS). J Neurosci Methods 2001;107:131–140.
Brunner G, Gabrilove J, Rifkin DB et al. Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan. J Cell Biol 1991;114:1275–1283.
Brunner G, Nguyen H, Gabrilove J et al. Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells. Blood 1993;81:631–638.
Oliver JA. Adenylate cyclase and protein kinase C mediate opposite actions on endothelial junctions. J Cell Physiol 1990;145:536–542.
Ding I, Huang K, Wang X et al. Radioprotection of hematopoietic tissue by fibroblast growth factors in fractionated radiation experiments. Acta Oncol 1997;36:337–340.
Zhang H, Wang JZ, Sun HY et al. The effects of GM1 and bFGF synergistically inducing adult rat bone marrow stromal cells to form neural progenitor cells and their differentiation. Chin J Traumatol 2004;7:3–6.(Daniel Tzu-bi Shiha,b, Do)