Effective Development of Hematopoietic Niche Cells From hESCs: hESC-Derived Stromal Cells Support LTC-ICs Better Than Bone Marrow-Derived Stromal Cells

Abstract 4804

Quantification of putative hematopoietic stem cells (HSCs) in human bone marrow or cord blood is typically assessed by its potential to mediate long-term, multilineage engraftment when transplanted into immunodeficient murine recipients. However, in vitro surrogate assays are extremely attractive due to their relative ease of implementation, low cost and improved throughput of results. The long-term culture initiating cell (LTC-IC) assay serves this purpose by quantifying the ability of putative HSCs in a given population to be cultured for an extended period, typically 5 weeks. Previously, we have derived both mesenchymal stromal cells (MSCs) and endothelial cells (ECs) from human embryonic stem cells (hESCs). MSCs and ECs are important components of the osteogenic and vascular hematopoietic niches and hESC-derived stromal cells can provide autologous supporting cell populations for putative hESC-derived HSCs. Therefore, we evaluated if hESC-derived MSCs (hESC-MSCs) and hESC-derived ECs (hESC-ECs) would have improved ability to support LTC-IC activity of UCB and hESC-derived hematopoietic cells compared to MSCs isolated from human bone marrow (BM-MSCs) and a murine stromal line, M2-10B4. In the present study, we utilized LTC-IC culture of both hESC-derived CD34⁺CD45⁺ populations and UCB 34⁺ cells to test their LTC-IC potential on hESC-MSCs, hESC-ECs and a combination of the two hESC-derived populations. LTC-IC frequency and maintenance of input populations was assessed by survival/proliferation and preservation of colony forming cell (CFC) progenitors over 5 weeks. Initial studies showed all stromal layers were able to support short-term (3 week) culture of CFCs from CD34⁺ UCB. Total cell proliferation was found to be greatest with the hESC-derived stroma at 3 weeks. Additionally, hESC-MSCs and hESC-ECs yielded CFC comparable to M2-10B4 and significantly more than BM-MSCs. Co-culture of CD34⁺ UCB with a 1:1 mixture of hESC-MSCs and hESC-ECs yielded similar proliferation and CFC results to either stroma alone. After 5 weeks of culture to quantify LTC-ICs, both of the hESC-derived cells again supported expansion of UCB total cell numbers better than BM-MSCs and similar to M2-10B4. LTC-IC yield at 5 weeks was 85 ± 29 and 74 ± 12 per 10³ input cells for hESC-MSC and hESC-EC, respectively, as compared to 15 ± 12 for BM-MSCs and 104 ± 56 for M2-10B4 co-culture. Combining the hESC-derived MSCs and ECs produced an LTC-IC yield of 54 ± 4. Next, we tested the ability of hESC-derived CD34⁺CD45⁺ cells to produce CFCs and LTC-ICs using co-culture with hESC-MSCs and M2-10B4. In short-term (2-3 week) assays, hESC-derived CD34⁺CD45⁺ cells declined in number to about 30% of the starting population on both stroma. However, between 3–5 weeks, cell number remained fairly constant on M2-10B4 while continuing to decrease on hESC-MSC. Interestingly, CFC yield for hESC-derived CD34⁺CD45⁺ cells at 2 weeks was 45 ± 43 per 10³ input cells on M2-10B4, while co-culture with hESC-MSCs regularly failed to produce any CFC at this time. Between 3–5 weeks of culture no CFC or LTC-ICs were produced from hESC CD34⁺CD45⁺ using any of the stromal cells. Similar results were seen for hESC-derived CD34⁺CD43⁺ populations. Together, these results have two key findings. First, hESC-derived ECs and MSCs are effective in supporting LTC-ICs from UCB, and do so better than human BM-MSCs. Second, these same stromal cells do not support survival of hESC-derived LTC-ICs. In this manner, these results closely mimic studies that show limited long-term hematopoietic engraftment of hESC-derived cells when transplanted into immunodeficient mice. Therefore, this system can now be used to aid in identification of both hematopoietic niche elements and specific cell populations that mediate development and support of putative HSCs derived from hESCs and iPSCs.