Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model

Abstract

Until now, ex vivo generation of CD34+ hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of non-human origin. While they provided invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system comprised of human bone marrow derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14+ monocyte-derived macrophages to generate CD34+ cells from hESCs in vitro. Human ESC-derived CD34+ cells generated using this method expressed surface makers associated with adult human HSCs and up-regulated hematopoietic stem cell genes compared to human bone marrow-derived CD34+ cells. Finally, transplantation of purified hESC-derived CD34+ cells into the pre-immune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multi-lineage hematopoietic activity with graft presence up to 16 weeks post-transplantation. This in vivo demonstration of engraftment and robust multi-lineage hematopoietic activity by hESC-derived CD34+ cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.

Novel engraftment and T cell differentiation of human hematopoietic cells inArt-/-IL2RG-/SCID pigs

“…As the field of biomedical SCID pig research expands, new techniques will arise to optimize human cell engraftment within SCID pig models. We can draw from previous large animal in utero injection protocols 41–44,53,58 , as well as humanization techniques performed in immunocompromised mice 59,60 . In our SCID pig model, we show that human T and B cells can develop.…”

Fetal articular cartilage regeneration versus adult fibrocartilaginous repair: secretome proteomics unravels molecular mechanisms in an ovine model

“…In addition, fetal sheep share many important physiological and developmental characteristics with humans and have proven themselves invaluable models for mammalian physiology ( Almeida-Porada et al, 2004 ; Jeanblanc et al, 2014 ). Results obtained in the fetal lamb have been directly applicable to the understanding of human fetal growth and development and are highly predictive of clinical outcome in a variety of applications including in utero stem-cell transplantation ( Almeida-Porada et al, 2004 , 2007 ; Jeanblanc et al, 2014 ; Kim et al, 2013 ; Kuypers et al, 2012 ; Liechty et al, 2000 ; Porada et al, 2005 ). A number of specific characteristics make sheep particularly well-suited for OA, regenerative medicine and fetal regeneration research enabling results of high clinical relevance to be obtained: (1) large size facilitating repeated sampling from individual animals and harvest of adequate sample sizes; (2) comparable body weight to humans; (3) similar mechanical exertion to humans ( Bruns et al, 2000 ; Russo et al, 2015 ); (4) relatively long life expectancy (lifespan 8-12 years) allowing longitudinal analysis as well as evaluation of long-term efficacy and safety of treatments; (5) long gestational period (150 days) provides sufficient temporal resolution to translate findings obtained in sheep into human parameters ( Jeanblanc et al, 2014 ); (6) extremely well-characterized immune development analogous to humans ( Almeida-Porada et al, 2004 ; Jeanblanc et al, 2014 ; Maddox et al, 1987 ; Miyasaka and Trnka, 1986 ; Osburn, 1981 ; Sawyer et al, 1978 ); (7) bone marrow ontogeny and niche development closely paralleling humans ( Jeanblanc et al, 2014 ).…”

Influence of a dual-injection regimen, plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model

“…Historically, mice, sheep, and man have undergone IUHSCT in the absence of MSCs or plerixafor, which resulted in low levels of engraftment (46). We recently utilized the transplantation regimen of Group 1 in studies to evaluate human embryonic stem cell derived CD34 + cell transplantation and reported engraftment in all of the recipients (47). …”