Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells

Abstract

Cell therapy using T cells has revolutionized medical care in recent years but limitations are associated with the difficulty of genome editing of the cells, the production of a sufficient number of cells and standardization of the product. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into T cells to provide a standardized homogenous product of defined origin in indefinite quantity, therefore they are of great potential to alleviate limitations of therapeutic T cell production. The differentiation of hPSCs takes place in two steps: first the induction of hematopoietic stem/progenitor cells (HSPCs), then the induction of lymphopoiesis by Notch signaling. However, the differentiation of T cells from hPSCs can be difficult and lack reproducibility. One parameter that needs to be better assessed is the potential of DLL1 vs. DLL4 ligands of the Notch pathway to induce T cells. In addition, culture of hPSCs is labor-intensive and not compatible with GMP production, especially when they are cultured on feeder cells. Thus, the definition of a robust GMP-compatible differentiation protocol from hPSCs cultured in feeder-free conditions would increase the accessibility to off-the-shelf hematopoietic and T cell progenitors derived from hPSCs. In this article, we describe an efficient, rapid and reproducible protocol for the generation of hematopoietic and T cell progenitors in two steps: (1) generation of HSPCs from embryoid bodies (EB) in serum free medium and GMP-compatible feeder-free systems, (2) directed differentiation of hPSC-derived HSPCs into T-cell progenitors in the presence of bone marrow stromal cells expressing Notch-ligands OP9-DLL1 vs. OP9-DLL4.

Generation of CD34+CD43+ hematopoietic progenitors to induce thymocytes from human pluripotent stem cells

“…Even if the signature of the cells after 25 days of co-culture on OP9-DLL1 was clearly typical of thymic progenitors at the DN3a stage, our culture conditions did not allow at that stage the acquisition of a functional TCR either in HSC-TD25 or in T-iPSC-Td25. As other, we observed in our differentiation some CD56 + cells (Flippe et al, 2020;Nishimura et al, 2013;Themeli et al, 2013).…”

“…We then subjected hPSCs derived from fibroblast or from T cells to HPSCs differentiation to better understand their respective potential and included hESCs as controls. The differentiation started by mesoderm induction, followed by hemogenic endothelium specification to finally yield CD34 + CD43 + HPSCs at day 9 (Figure 2A) (Flippe et al, 2020). Overall, all pluripotent cell lines differentiated efficiently, yielding in average 37% of CD34 + cells, although we observed a more efficient generation of CD34 + cells using the T04.01B clone vs. the other clones (Supplementary figure 1A).…”

“…Furthermore, it has been demonstrated that T cell reprogramming can increase the lifespan of cells because newly produced cells have longer telomeres, which represents a significant therapeutic advantage (Nishimura et al, 2013). To efficiently direct the differentiation of hiPSCs to the hematopoietic lineage, we used our optimized feeder-free protocol (Flippe et al, 2020). After 9 days of differentiation, we obtained CD34 + CD45 + cells regardless of the hiPSCs or hESCs lines used.…”

“…We then subjected HPSCs and CD34 + cord blood HSCs to T cell differentiation using DLL1 or DLL4-overexpressing OP9 cells co-culture. Of note, we recently defined that OP9-DLL1 and OP9-DLL4 were equivalently potent to induce T cell lineage (Flippe et al, 2020). Briefly, D9 EBs were dissociated and seeded on DLL1/4-OP9 cells in a medium supplemented with SCF, FLT3l and IL-7 (Td0) and analyzed after 15 (Td15), 20 (Td20) and 25 days (Td25) (Figure 2A).…”

Effects of somatic mutations on cellular differentiation in iPSC models of neurodevelopment

“…However, in vitro differentiation is subject to substantial technical and biological confounders that often lead to variable differentiation outcomes, a challenge to scaling and interpreting results from these models of disease 1 . The underlying reasons for this variability are not well understood, but different factors have been proposed: protocol optimisation 2 , culture maintenance 3 , passage number 4 , molecular determinants 5 , inter-laboratory variation 6 , cell line intrinsic properties 7 or the loss of iPSC heterogeneity in culture 8 .…”