CD34+ Cells Derived from Human Embryonic Stem Cells Demonstrate Hematopoietic Stem Cell Potential In Vitro and in Vivo.
Authors: Kaufman Dan S. , Woll Petter S. , Martin Colin H. , Linehan Jon L. , Tian Xinghui 
Published: October 2019

Abstract
We have previously described methods to use either stromal cell co-culture or embryoid-body (EB) formation to support the hematopoietic development of undifferentiated human ES cells (both H1 and H9 cell lines). Either FBS-based media or serum-free media with specific cytokines can be used to derive CD34+ cells, CD45+ cells and hematopoietic progenitors as identified by colony-forming cell (CFC) assays that give rise to mature myeloid, erythroid, and megakaryocytic cells. Genes such as RUNX1, HOXB4, TAL1, and GATA2, all known to be expressed during early hematopoiesis are up-regulated during hematopoietic differentiation of human ES cells. Here, we advance these studies to demonstrate that human ES cell-derived CD34+ cells function as early hematopoietic precursors in surrogate hematopoietic stem cell (HSC) assays. The long-term culture initiating cell (LTC-IC) assay is commonly used to quantify hematopoietic precursors that can be maintained in culture for 5 or more weeks. Human cord blood (CB)-derived CD34+ cells have a LTC-IC frequency of approximately 1:30. We demonstrate LTC-ICs can also be identified from human ES cell-derived CD34+ at a frequency of approximately 1:400. These results suggest CD34+ cells from human ES cells are more heterogeneous than CD34+ cells from CB. Furthermore, we now demonstrate in vitro culture of human ES cell-derived CD34+ cells identify these cells as lymphocyte precursors. Here, we used a natural killer (NK) cell-initiating cell assay (NK-IC) where CD34+ cells are cultured on AFT024 stromal cells in media containing IL15, IL7, and other defined cytokines for 2–4 weeks. Under these conditions, both CB and human ES cell-derived cells give rise to lymphoid cells (NK cells) with over 40% CD45+CD56+ cells. Under alternative culture conditions, CD3+ T cells can also be produced from CD34+ human ES cell-derived cells. Therefore, CD34+ cells derived from human ES cells represent both myeloid and lymphoid precursor cells. Since it is not possible to define a HSC population based solely on in vitro assays, we have examined the potential for human ES cell-derived hematopoietic cells to engraft in sublethally irradiated NOD/SCID mice. Detection of scid-repopulating cells (SRCs) are considered a better surrogate for HSCs. Bone marrow, peripheral blood, and splenocytes were examined for human CD34+ and CD45+ cells 3–6 months after injection of human ES cell-derived blood cells. PCR for human chromosome 17-specific alpha-satellite DNA was also done to confirm the presence of human cells in all mice showing evidence of engraftment. We consistently find stable engraftment with 0.5–3% human CD45+ cells in the bone marrow of these mice. To better define these cells as HSCs, secondary transplants also demonstrate stable engraftment. Importantly, no teratomas are demonstrated in mice injected with differentiated human ES cells. These results demonstrate that HSCs with long-term engraftment and multi-lineage potential can be routinely and efficiently generated from human ES cells.