Characterization of autologous mesenchymal stem cell-derived neural progenitors as a feasible source of stem cells for central nervous system applications in multiple sclerosis

Abstract

Bone marrow mesenchymal stem cell-derived neural progenitors (MSC-NPs) are a potential therapeutic source of cells that have been shown to be efficacious in a preclinical model of multiple sclerosis (MS). To examine the feasibility of using MSC-NPs as an autologous source of cells to promote central nervous system (CNS) repair in MS, this study characterized human MSC-NPs from a panel of both MS and non-MS donors. Expanded MSCs showed similar characteristics in terms of growth and cell surface phenotype, regardless of the donor disease status. MSC-NPs derived from all MSCs showed a consistent pattern of gene expression changes that correlated with neural commitment and increased homogeneity. Furthermore, the reduced expression of mesodermal markers and reduced capacity for adipogenic or osteogenic differentiation in MSC-NPs compared with MSCs suggested that MSC-NPs have reduced potential of unwanted mesodermal differentiation upon CNS transplantation. The immunoregulatory function of MSC-NPs was similar to that of MSCs in their ability to suppress T-cell proliferation and to promote expansion of FoxP3-positive T regulatory cells in vitro. In addition, MSC-NPs promoted oligodendroglial differentiation from brain-derived neural stem cells that correlated with the secretion of bioactive factors. Our results provide a set of identity characteristics for autologous MSC-NPs and suggest that the in vitro immunoregulatory and trophic properties of these cells may have therapeutic value in the treatment of MS.

Figure 1.

Growth kinetics and cell surface phenotype of MSCs derived from bone marrow of healthy controls and from MS patients. (A): Cumulative population doublings of MSCs from control and MS patients. MSCs were derived from MS patient donors (n = 10) and from non-MS controls (n = 4). (B): Cumulative population doublings of MSCs derived from MS patient donors (n = 3) cultured in either 10% FBS or 10% AS for four passages. (C, D): Cell surface phenotype of representative samples of healthy control MSCs (7071L) (C) and MS patient-derived MSCs (MS-066) (D). Cell surface staining was carried out using fluorescein isothiocyanate- or phosphatidylethanolamine-conjugated antibodies against CD90, CD105, CD73, CD45, CD34, CD14, CD19, and HLA-DR (black histograms) and compared with isotype controls (gray histograms). Numbers indicate the mean fluorescence intensity expressed as fold increase above the isotype control for each antibody. Abbreviations: AS, autologous serum; CPD, cumulative population doublings; FBS, fetal bovine serum; MS, multiple sclerosis; MSC, mesenchymal stem cell.

Figure 2.

Characterization of MSC-NP morphology and gene expression. (A): Spindle-shaped morphology of human MSCs viewed by light microscopy at a magnification of ×10. (B): Spherical morphology of MSC-NPs after 3 weeks of neural induction. The cells were viewed by light microscopy at a magnification of ×20. Inset: Nestin immunofluorescence (red) of MSC-NP neurosphere showing that the majority of 4′,6-diamidino-2-phenylindole-positive cells (blue) expressed Nestin. The cells were viewed by fluorescence microscopy at a magnification of ×20. (C): Time course of gene expression changes upon MSC-NP induction. MSC-NPs, compared with MSCs, showed increased mRNA expression of neural markers Nestin, NF-M, GFAP, and CXCR4 and decreased expression of MSC markers CD90 and SMA. The values represent the means ± SE of four separate MSC donors. (D): Panel of gene expression changes in MSC-NPs derived from control (n = 4) or MS (n = 10) patients. Abbreviations: GFAP, glial fibrillary acidic protein; MS, multiple sclerosis; MSC, mesenchymal stem cell; MSC-NP, mesenchymal stem cell-derived neural progenitor; NF-M, Neurofilament-M; SMA, α-actin smooth muscle isoform.

Figure 3.

Expression of neural and mesenchymal protein markers in MSC-NPs. (A): Immunocytochemistry demonstrating qualitative changes in Nestin, NF-M, GFAP, CXCR4, SMA, and CD90 in MSC-NPs compared with MSCs. A representative experiment using MS patient-derived cells is shown. (B): Flow cytometry analysis of protein expression in MSC and MSC-NP populations. MSCs (blue histograms) or MSC-NPs (red histograms) were labeled with antibodies against the indicated markers or with isotype controls (black histograms), followed by detection with Alexa 488-conjugated secondary antibody. Numbers indicate the mean fluorescence intensity expressed as fold increase above the isotype control for each antibody. A representative experiment is shown. Abbreviations: GFAP, glial fibrillary acidic protein; MSC, mesenchymal stem cell; MSC-NP, mesenchymal stem cell-derived neural progenitor; NF-M, Neurofilament-M; SMA, α-actin smooth muscle isoform.

Figure 4.

MSC-NPs lose mesodermal differentiation capacity. (A): Alizarin red staining of calcium deposition after osteogenic differentiation demonstrating limited osteogenic differentiation by MSC-NPs compared with MSCs. (B): Oil Red O staining of accumulated lipids after adipogenic differentiation demonstrating limited adipogenic differentiation by MSC-NPs compared with MSCs. (C): Total calcium deposition was measured by calcium (CPC) Liquicolor assay after osteogenic differentiation of MSCs or MSC-NPs. The cells were cultured in MSC growth medium alone as a control. The data shown are representative of three separate experiments. (D): Quantitative real-time polymerase chain reaction of ALPL and IBSP mRNA levels after osteogenic differentiation of MSCs or MSC-NPs. (E): ADIPOQ and PPARγ mRNA levels after adipogenic differentiation of MSCs or MSC-NPs. All values represent the averages of two independent experiments using both control and MS-derived cells, and the error bars represent SD. *, p < .05 and demonstrates significance compared with MSCs. Abbreviations: ADIPOQ, adiponectin; ALPL, alkaline phosphatase; IBSP, integrin-binding sialoprotein; MSC, mesenchymal stem cell; MSC-NP, mesenchymal stem cell-derived neural progenitor; PPARγ, peroxisome proliferator-activated receptor γ.

Figure 5.

Immunoregulatory properties of MSC-NPs and MSCs are similar. (A): Proliferation of phytohemagglutinin-stimulated CFSE-labeled CD4⁺ T cells (white histogram) is reduced in the presence of cocultured MSC-NPs (black filled histogram) as indicated by a right shift in CFSE fluorescence. (B): Reduced proliferation shown in (A) as measured by increased MFI of CFSE in cocultured T cells (TC+MSC-NP or TC+MSC) compared with T cells alone (TC). MSCs or MSC-NPs were derived from cont or MS donors as shown. (C, D): Coculture of CD3/CD28-stimulated naïve CD4⁺ T cells with MSC-NPs or MSCs promotes FoxP3⁺ T regulatory cells measured by flow cytometry (C) and by FoxP3 expression by quantitative polymerase chain reaction (D). (E): Levels of immunoregulatory factors (TNFα, IL-12, IFNγ, TGFβ, and IL-6) secreted in cocultures from (C) and determined by Luminex assay. MSCs and MSC-NPs in (D, E) were derived from MS donors. The values are shown relative to T cells alone. In all panels, the values represent the averages of at least two independent experiments, and the error bars represent SD. *, p < .05; **, p < .01, demonstrating significance compared with TC control. Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; cont, control non-multiple sclerosis; IFNγ, interferon γ; IL, interleukin; MFI, mean fluorescence intensity; MS, multiple sclerosis; MSC, mesenchymal stem cell; MSC-NP, mesenchymal stem cell-derived neural progenitor; TC, T cell; TGFβ, transforming growth factor β; TNFα, tumor necrosis factor α.

Figure 6.

Trophic effects of MSC-NPs on the oligodendroglial differentiation of rat brain-derived neural stem cells. (A–H): Immunocytochemical analysis of rNSCs alone (A–D) or cocultured with human MSC-NPs (E–H) after 2 weeks of differentiation. Oligodendrocytes were detected by GalC (A, E) (green) and MBP (B, F) (green), astrocytes by GFAP (C, G), and neurons by β3-tub (D, H) (green). Human MSC-NPs in (E–H) were detected by staining for human nuclear antigen (hNA; red). All nuclei were stained by DAPI (blue). One representative experiment is shown using cells derived from an multiple sclerosis (MS) donor. (I): Quantification of immunostaining compiled from two separate experiments using MSC-NPs derived from control and MS donors as shown. Quantification represents positive staining normalized for the number of DAPI-positive, hNA-negative cells. (J): Quantitative polymerase chain reaction of PLP, GFAP, and β3-tubulin mRNA expressed in MSC-NP/rNSC cocultures relative to rNSCs alone. (K): Levels of secreted factors in rNSC/MSC-NP cocultures, or rNSCs or MSC-NPs alone after culturing for 2 weeks under differentiating conditions. Growth factors were measured by Luminex and enzyme-linked immunosorbent assay. (I, J): The values represent the averages of three separate experiments, and the error bars represent SD. *, p < .05; **, p < .01, demonstrating significance compared with rNSC control. Abbreviations: β3-tub, β3-tubulin; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; cont, control; DAPI, 4′,6-diamidino-2-phenylindole; GalC, galactocerebroside; GDNF, glial-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; HGF, hepatocyte growth factor; IGF1, insulin-like growth factor 1; IL-11, interleukin 11; MBP, myelin basic protein; MS, multiple sclerosis; MSC, mesenchymal stem cell; MSC-NP, mesenchymal stem cell-derived neural progenitor; PLP, proteolipid protein; rNSC, rat neural stem/progenitor cell; SDF1, stromal cell-derived factor 1; TGFβ, transforming growth factor β; VEGF, vascular endothelial growth factor.

Figure 7.

Expression of candidate trophic and immunomodulatory factors is increased in mesenchymal stem cell-derived neural progenitors (MSC-NPs) compared with MSCs. (A): Gene expression of candidate trophic factors bFGF, GDNF, HGF, IGF1, IL-11, SDF1, TGFβ, and VEGF-A was determined by quantitative real-time polymerase chain reaction (Q-RT-PCR) in MSC-NPs derived from control (n = 4) and MS (n = 10) and expressed as fold increase in mRNA levels relative to MSCs from which they were derived. The dotted line represents a value of 1 and represents a lack of gene expression change between MSCs and MSC-NPs. (B): Gene expression of immunomodulatory factors CXCL10, IDO, IL-6, IL-10, TLR2, TLR3, and TLR4 was determined by Q-RT-PCR in MSC-NPs derived from control (n = 4) and MS (n = 10) and expressed as fold increase in mRNA levels relative to the MSCs from which they were derived. Abbreviations: bFGF, basic fibroblast growth factor; GDNF, glial-derived neurotrophic factor; HGF, hepatocyte growth factor; IDO, indoleamine-2,3-dioxygenase; IGF1, insulin-like growth factor 1; IL, interleukin; MS, multiple sclerosis; SDF1, stromal cell-derived factor 1; TGFβ, transforming growth factor β; TLR, Toll-like receptor; VEGF-A, vascular endothelial growth factor-A.