Retinoic Acid Regulates Hematopoietic Development from Human Pluripotent Stem Cells

Abstract

SummaryThe functions of retinoic acid (RA), a potent morphogen with crucial roles in embryogenesis including developmental hematopoiesis, have not been thoroughly investigated in the human setting. Using an in vitro model of human hematopoietic development, we evaluated the effects of RA signaling on the development of blood and on generated hematopoietic progenitors. Decreased RA signaling increases the generation of cells with a hematopoietic stem cell (HSC)-like phenotype, capable of differentiation into myeloid and lymphoid lineages, through two separate mechanisms: by increasing the commitment of pluripotent stem cells toward the hematopoietic lineage during the developmental process and by decreasing the differentiation of generated blood progenitors. Our results demonstrate that controlled low-level RA signaling is a requirement in human blood development, and we propose a new interpretation of RA as a regulatory factor, where appropriate control of RA signaling enables increased generation of hematopoietic progenitor cells from pluripotent stem cells in vitro.

Antagonism of PPAR-γ signaling expands human hematopoietic stem and progenitor cells by enhancing glycolysis

“…Treatment of CB CD34 + cells with GW9662 resulted in downregulation of a number of differentiation associated genes 20–25 , including CD38 , CD1d , HIC1 , FAM20C , DUSP4 , DHRS3 and ALDH1A2 (Fig. 3a,b and Supplementary Fig.7a,b), suggesting that PPARγ antagonism may maintain stemness of CB CD34 + cells, at least in part by preventing differentiation.…”

Reactive Oxygen Species Impair the Function of CD90+ Hematopoietic Progenitors Generated from Human Pluripotent Stem Cells

“…Since elevated ROS has been shown to impair the function of both murine and human HSCs, and progenitors, and since these primary cells, when cultured in standard in vitro culture conditions, rapidly shift from a ROS lo to a ROS hi state , we assessed the levels of ROS in our pluripotent stem cell differentiation system, previously described , and evaluated the impact of ROS on the in vitro generation and functionality of hematopoietic cells. Following 16 days of differentiation from the pluripotent state, hematopoietic cell fractions were identified by their cell surface phenotype using the established markers, as follows: total hematopoietic cell fraction identified as CD43/45 + (combined use of early‐ & pan‐hematopoietic markers) , the hematopoietic progenitor fraction as CD43/45 + CD34 + , and our more primitive hematopoietic cell fraction, previously described as HSC‐like , as CD43/45 + CD34 + CD90 + . The cell permeable dye CellROX Deep Red, becoming fluorescent upon presence of intracellular ROS, was used to measure ROS in all cell populations.…”

“…Given that hPSC‐derived hematopoietic cells have persistently demonstrated proliferative capacity deficits compared to adult and neonatal counterparts (bone marrow and cord blood progenitors), we hypothesized that hPSC‐derived hematopoietic cells are functionally impaired as a direct result of the elevated ROS levels and its associated consequences to cell proliferation, and survival. In this study, using our previously established protocol for generating hPSC‐derived hematopoietic progenitors , we demonstrate that the vast majority of hematopoietic progenitors generated during in vitro hPSC‐to‐blood differentiation possess high levels of intracellular ROS as compared to noncultured cord blood progenitors. Functional evaluation of hPSC‐derived hematopoietic progenitors revealed that ROS hi cells have higher levels of DNA damage and reduced colony forming capacity as compared to ROS lo cells.…”

“…Indeed, while we observe a significant difference in DNA damage for ROS lo hPSC‐derived hematopoietic progenitors as compared to their ROS hi counterparts, there is still a significant difference in the extent of DNA damage of these cells as compared to ROS lo cells from noncultured cord blood, indicating that further improvements are required if ROS is to be further normalized toward physiological levels. While it is possible to generate hematopoietic cells that are similar to functional HSCs in terms of cell surface markers, as well as in their definitive myeloid/lymphoid differentiation capacity, the hPSC‐derived cells lack functional engraftment capacity . It is often assumed that there is a maturation or development signal missing in in vitro differentiation systems that prevents the generation of robust repopulating HSCs.…”

“…Although largely quiescent, these cells are sensitive to genotoxic insult and upon re‐entry into cell cycle are vulnerable to genotoxicity responses aimed at preventing cancer cell development . With the goal of understanding human hematopoietic development and ultimately being able to provide an alternative source of HSCs for therapeutic purposes, progress has been made to generate transplantable hematopoietic cells in vitro by directed differentiation of human pluripotent stem cells (hPSCs) and direct reprogramming to the hematopoietic lineages . However, achieving robust engraftment of in vitro generated human HSCs has been extremely difficult.…”