iPSC-derived mesenchymal stromal cells are less supportive than primary MSCs for co-culture of hematopoietic progenitor cells

Abstract

In vitro culture of hematopoietic stem and progenitor cells (HPCs) is supported by a suitable cellular microenvironment, such as mesenchymal stromal cells (MSCs)-but MSCs are heterogeneous and poorly defined. In this study, we analyzed whether MSCs derived from induced pluripotent stem cells (iPS-MSCs) provide a suitable cellular feeder layer too. iPS-MSCs clearly supported proliferation of HPCs, maintenance of a primitive immunophenotype (CD34(+), CD133(+), CD38(-)), and colony-forming unit (CFU) potential of CD34(+) HPCs. However, particularly long-term culture-initiating cell (LTC-IC) frequency was lower with iPS-MSCs as compared to primary MSCs. Relevant genes for cell-cell interaction were overall expressed at similar level in MSCs and iPS-MSCs, whereas VCAM1 was less expressed in the latter. In conclusion, our iPS-MSCs support in vitro culture of HPCs; however, under the current differentiation and culture conditions, they are less suitable than primary MSCs from bone marrow.

Fig. 1

The hematopoietic supportive function of iPS-MSCs. a CD34⁺ cells were stained with CFSE and cultured with or without feeder cells for 5 days. Co-culture of HPCs with either MSCs or iPS-MSCs enhanced the number of cell divisions significantly (**P < 0.01; ***P < 0.001; n = 5—each with three biological replicates for MSCs and iPS-MSCs; MFI = mean fluorescence intensity). b Dot plots show CD34 expression in relation to the number of cell divisions (as a reference we used additional measurements at day 0; the number of cell divisions in different CFSE-gates is indicated). c We gated for specific cell division numbers and analyzed the signal intensity of CD34, CD133, CD45, and CD38 as compared to culture without feeder layer (gray). Co-culture with MSCs (red) or iPS-MSCs (blue) led to an increase of CD34 and CD133 expression and a decrease of CD45 expression in proliferating cells (without feeder vs. MSCs: *P < 0.05, **P < 0.01; without feeder vs. iPS-MSCs: ^# P < 0.05, ^## P < 0.01; MSCs vs. iPS-MSCs : ^§ P < 0.05; n = 5—each with three biological replicates for MSCs and iPS-MSCs). d CFU frequency was significantly increased by co-culture with either MSCs or iPS-MSCs (**P < 0.01, n = 3—each with three biological replicates for MSCs and iPS-MSCs). There was no significant bias for specific types of colonies. BFU-E = burst-forming-unit erythroid; CFU-E = colony-forming-unit erythroid; CFU-G = colony-forming-unit granulocyte; CFU-M = colony-forming-unit macrophage; CFU-GM = colony-forming-unit granulocyte, macrophage; CFU-GEMM = colony-forming-unit granulocyte, erythrocyte, macrophage, megakaryocyte. e Frequency of long-term culture initiating cells (LTC-IC) was significantly higher in co-culture with primary bone marrow-derived MSCs as compared to iPS-MSCs or without stromal support (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3—each with three biological replicates for MSCs and iPS-MSCs). Mean ± S.D. is depicted

Fig. 2

Differences in cell-cell interaction with different feeder layers. a The percentage of HPCs with elongated morphology was scored as described before [6]. Particularly, co-culture with MSCs stimulated cellular elongation (*P < 0.05, n = 3—each with three biological replicates for MSCs and iPS-MSCs). b Gene expression of relevant genes for cellular interaction with HPCs were analyzed in MSCs, iPSCs, and iPS-MSCs. Overall, all genes were expressed at very similar levels in primary MSCs and iPS-MSCs—except for Laminin β1 (LAMB1; higher expressed in iPS-MSCs; **P < 0.01) and vascular cell adhesion molecule 1 (VCAM1; higher expressed in MSCs; **P < 0.01). c Mean fluorescence intensity of VCAM1 (CD106) expression in flow cytometric analysis (three biological replicates for MSCs and iPS-MSCs). Mean ± S.D. is depicted