Human pluripotent stem cells (hPSCs), when exposed to the right molecular signals in the right order, can become any cell type in the human body. Regenerative medicine seeks to derive blood-forming stem cells (hematopoietic stem cells, HSCs) from hPSCs, since this would preclude the need for bone marrow donors in life-saving HSC transplantations.

A study led by researchers from Mount Sinai and the San Raffaele Telethon Institute for Gene Therapy in Milan, has uncovered a key component of the developmental program that produces precursors to HSCs in the human embryo.

“We anticipate this methodology will provide a useful platform for developmental hematopoiesis and disease modeling studies, and in the pursuit of the in vitro generation of therapeutically relevant hematopoietic cells,” the authors noted.

The findings were published in the journal Nature Cell Biology, in an article titled, “Identification of a retinoic acid-dependent hemogenic endothelial progenitor from human pluripotent stem cells.” The new method described in the study brings scientists a step closer to developing HSCs and specialized blood cell types in tissue culture.

Christopher Sturgeon, PhD, associate professor at Mount Sinai, is the senior author of the paper.

Senior author of the study, Christopher Sturgeon, PhD, associate professor of cell, developmental, and regenerative biology and medicine, hematology, and medical oncology at the Black Family Stem Cell Institute at the Icahn School of Medicine at Mount Sinai said, “Over the years, many powerful techniques have been developed to generate blood progenitors from pluripotent stem cells. However, in the absence of transgene expression strategies, these approaches would not yield transplantable hematopoietic stem cells. So, we thought we could look into how embryos make these cells.”

During embryonic development, HSCs are produced from specialized mesodermal cells in the embryo called the hemogenic endothelium (HE) upon exposure to the signaling molecules notch and retinoic acid. In earlier studies, researchers have obtained HE from hPSCs, but these HE cells do not produce HSCs. Current methods for deriving blood progenitors from hPSCs do so in the absence of retinoic acid. Identifying a way to obtain blood progenitors that require retinoic acid for their development is a critical first step to deriving HSCs in tissue culture.

“Multiple studies have established that during vertebrate embryonic development, the retinoic acid signaling pathway is required for the development of HSCs. Critically, all the strategies that have been described to date, to differentiate pluripotent stem cells into blood cells employ defined media conditions that lack retinoic acid, meaning that the progenitors being obtained were clearly developing from a retinoic acid-independent program, and could explain why they failed to yield hematopoietic stem cells,” said Sturgeon.

Earlier studies have obtained HE expressing a patterning gene called HOXA and requiring a developmental signal called Wnt. However, this definitive HE is unresponsive to retinoic acid. The new study shows Wnt-dependent mesodermal cells are composed of two populations before the HE is specified: mesodermal cells that do not express CXCR4 but do express CYP26A1 generate HOXA expressing HE without retinoic acid while mesodermal cells expressing CXCR4 and ALDH1A2  generate HOXA expressing HE that requires retinoic acid.

Sturgeon said, “Here, we identified for the first time, a progenitor population that positively responds to retinoic acid to give rise to blood progenitors. Remarkably, these responsive cells are at a developmental stage equivalent to the very early human embryo—around 19 days of gestation—which is much earlier than previously assumed based on mouse developmental studies. Now we can focus on how to properly guide, with additional signals, the maturation of these cells into a blood-forming stem cell.”

The authors also showed that both retinoic acid independent and dependent HE contain sets of RNA messages that are found in distinct populations in the early human embryo, including HE cells in the human embryo that form HSCs. Their analysis found that stem cell populations derived from hPSCs were transcriptionally similar to cells in the early human embryo.

“This revised model of human hematopoietic development provides essential resolution to the regulation and origins of the multiple waves of hematopoiesis. These insights provide the basis for the generation of specific hematopoietic populations, including the de novo specification of HSCs,” the authors noted.

In this study, researchers examined the dependence on retinoic acid in early cell types derived from hPSCs. The scientists performed single-cell RNA-seq on day 3 of differentiation cultures to understand the transcriptomic signatures of mesodermal populations obtained during early differentiation. The team developed a new strategy to obtain cells that are transcriptionally similar to HE cells found in the human embryo by stimulating a discrete population of cells with retinoic acid.

“Our detailed gene expression analyses during early stages in this process identified that a population of cells poised to respond to retinoic acid was always present, but we were not providing that signal at the appropriate stage. Now, we have identified the stage-specific requirements for retinoic acid in this process,” said Sturgeon.

Leonard Zon, MD, a professor of pediatric medicine at Harvard Medical School, investigator at Howard Hughes Medical Institute, and director of the stem cell program at Children’s Hospital Boston said, “This is an excellent article in which the investigators define specific blood cell populations during human iPSC differentiation that are retinoic acid responsive.  These cells can be transplanted and can engraft immunodeficient mice.  This is an important step in the ultimate generation of blood stem cells from pluripotent cells.” Zon was not involved in the current study.

In future studies, Sturgeon and his team plans to better understand additional signals required for these progenitors to give rise to bona fide hematopoietic stem cells.