Figure 1.
A schematic diagram representing the order of differentiation of the cells used in this study.
Table 1.
Description of cell lines.
Figure 2.
Expression of HLA-A and HLA-B in hESC and hMSC.
Representative flow histograms showing HLA-A and -B expression in stem cell lines using allele-specific antibodies (anti-A2, -A3, -B7, and -B13). (A) In separate experiments, hESC lines (huES9 and KMEB2) showed a low expression of HLA-A alleles (A2 and A3, respectively) and no expression of the HLA-B alleles (B13 and B7, respectively). IFNγ stimulation up-regulated HLA-A alleles and induced of modest expression of HLA-B alleles. (B) Panel B shows representative flow histograms of an hMSC cell line (ToB11-13) both un-stimulated and IFNγ stimulated (72 hr) demonstrating a high constitutive expression of HLA-A2 and a relatively low expression of HLA-B7. Both antigens were up-regulated after stimulation with IFNγ. Panel (C) compares the expression of HLA-A and HLA-B between hESC (huES9) and hMSC (ToB11-13) during basal, un-induced conditions and after stimulation for 72 h with 25 ng/µl IFNγ. HLA expression was measured by flowcytometry as molecules of equivalent fluorochromes (MEF).
Figure 3.
Differentiation of hMSC to adipocytes and osteoblasts.
Representative images for Oil O Red and Alizarin red staining demonstrating adipocyte and osteoblasts differentiation of hMSC cell lines cultured under adipogenic and osteogenic conditions, respectively. Non-differentiated cells were not stained (A and D), while the adipocyte differentiated cells were stained with Oil O red (B) and Alizarin red stained the matrix formed by osteoblasts (E). qPCR data shows marked up-regulation of PPAR gamma gene expression after adipocyte differentiation (C) and up-regulation of Collagen 1 gene expression in osteoblast differentiated cells (F).
Figure 4.
Expression of HLA-A and HLA-B on hMSC and in their differentiated adipocyte and osteoblast progenies.
Locus-specific HLA antigen expression before and after 13 days of differentiation of hMSC to adipocytes (A), or 16 days of differentiation to osteoblasts (B) in one hMSC-Tert4 cell line and three primary hMSC cell lines (Table 1). The analyzed HLA-A alleles were HLA-A2 (N = 3) and -A3 (N = 2). The analyzed HLA-B alleles were HLA-B7 (N = 3), -B27 (N = 1) and -B8 (N = 1) (data pooled according to locus, each data point represents mean value for 3 experiments for each cell line).
Figure 5.
Cell surface HLA expression of hHSC and lymphocytes derived from BM and peripheral blood.
All allelic forms of HLA studied were highly expressed in all the cell types shown. However, HLA-A2 was expressed at significantly higher levels than HLA-B7 and -B8 on hHSC, BM CD4+ T cells, and BM CD8+ T cells. When BM lymphocytes and peripheral blood lymphocytes were compared, there was a marginal (but non-significant) difference in the expression of HLA-A2 and -B8 alleles which tended to have higher expression in peripheral blood than in BM. A significantly higher expression level of HLA-B7 was observed on peripheral blood CD4+ and CD8+ T lymphocytes. In peripheral blood, the expression of HLA-B8 was significantly lower than HLA-A2 and -B7 on CD4+ and CD8+ T lymphocytes. Peripheral CD19+ lymphocytes expressed similar amounts of HLA-A2, -B7 and -B8.