Human Embryonic Stem Cell-Derived CD34+ Cells Preferentially Develop into Endothelial Cells When Transplanted into Neonatal Immunodeficient Mice
Authors: Tian Xinghui , Hexum Melinda K. , Penchev Vesselin R. , Taylor Russell J. , Shultz Leonard , Kaufman Dan S. 
Published: October 2019

Abstract
Selection for CD34+ cells from human bone marrow, umbilical cord blood and mobilized peripheral blood cells has been used to enrich for putative hematopoietic stem cells that are typically characterized by the ability to provide long-term multi-lineage engraftment after transplantation into NOD/SCID mice. In vitro studies have shown that CD34+ cells derived from human embryonic stem cells (hESC) have the similar capacity to develop into diverse mature blood cell lineages under defined conditions, though they may represent an even more primitive progenitor population with bi-potent ability to developed into both hematopoietic and endothelial cells. Previously, we and others have demonstrated in vivo hematopoietic engraftment of an unsorted heterogeneous population of differentiated hESC-derived cells injected into NOD/SCID mice. Here, to determine whether sorted CD34+ cells isolated from differentiated hESC have the equivalent SCID-repopulating potential, we directly delivered the CD34+ cells into the liver of neonatal NOD/SCID/gamma c (null) mice. Stable expression of firefly luciferase (luc) in the hESC derived-CD34+ cells allowed us to non-invasively track the engraftment patterns by in vivo bioluminescent imaging. Luc+ CD34+ cells were produced by co-culture of hESC with M2-10B4 mouse bone marrow-derived stromal cells. Each mouse (n=18) was injected with 2.5 – 9.4 × 10^5 CD34+ cells isolated at an early stage of differentiation (day 6–9 of co-culture). Successful injections were routinely confirmed by the bioluminescent signal detected in the liver at 1 week post-transplantation. There was typically a decrease of luc signal observed between 2–5 weeks post-transplantation, followed by the stabilizing and increasing of luc-expressing cells at later time points. Dramatic increase of bioluminescent signal in the livers was routinely demonstrated 8–12 weeks post-transplantation. The bioluminescence foci were initially maintained in the liver. Migration of luc+ cells from liver to bone marrow, spleen, kidney and mediastinum/thymus was also detected at later time points (10+ weeks post-transplantation). Flow cytometric analysis demonstrated a significant population of human HLA-class I+ cells (0.1–5.35%) in the engrafted livers (11 out of 15 mice), with the majority of the human CD34+CD31+ endothelial cells (0.03–4.78%). In most of cases, these endothelial cells also co-expressed CD73. No human CD45+ cells were found in any of the tissues evaluated. This is in contrast to CD34+ cells obtained from human cord blood utilized as a positive control cell populations that routinely demonstrated high levels of CD45+ cells (86.92±10.5%) of diverse hematopoietic lineages in the liver when transplanted in this model. Immunohistochemical staining showed that the hESC-derived CD34+CD31+ cells localized in vessels of the engrafted liver. Remarkably, the hESC-derived luc+CD34+CD31+ cells could be isolated several months post-transplant and re-cultured to clearly demonstrate typical endothelial cell characteristics including expression of vonWillebrand factor and VE-Cadherin, taking up of ac-LDL, and formation of capillary-like tubes when cultured in Matrigel. Additionally, ex vivo imaging of the exercised spleens and their surrounding connective tissues demonstrated luc+ cells located in the surrounding connective/mesenchymal tissues. Similar luc+ mesenchymal tissues were also found around the kidneys. Importantly, no visible teratomas were found in any of the transplanted mice. Together, these results demonstrate hESC-derived CD34+ cells preferentially develop into endothelial cells after transplantation into the fetal liver environment, with some additional development of mesenchymal tissues, but minimal hematopoietic cells. These studies demonstrate hESC-derived CD34+ cells are suitable for in vivo models to correct of vascular disease and further studies are underway to define signals required for improved hematopoietic engraftment of purified hESC-derived CD34+ cells.