Reaching the Holy Grail: Making Hematopoietic Stem Cells in a Dish

Generating long-term engrafting hematopoietic stem cells (HSCs) from human-induced pluripotent stem cells (hiPSCs) has been a long-standing goal in regenerative medicine and has been seen as the holy grail. The ability to produce patient-specific HSCs would revolutionize treatments for a range of hematological disorders, including leukemia, anemia, and immunodeficiencies, by circumventing the limitations of donor availability and immune rejection. In addition, this would provide a unique, renewable platform for the study of both physiological and diseased hematopoietic processes.

Historically, the study of embryonic development has provided instructions to make blood cells in vitro from hiPSCs (Ditadi et al., 2017). However, understanding what kind of blood cells are made in a dish is challenging as developmental hematopoiesis in vertebrates, including in humans, unfolds through multiple temporal waves and within specific anatomical niches (Ditadi et al., 2017). This layered architecture yields a mixture of differentiated blood cells to meet the needs of the growing embryo, progenitors with restricted potential, and HSCs that sustain lifelong hematopoiesis. Previous studies have highlighted that each program can be traced to distinct mesodermal cell populations, which can be selectively derived from hiPSC using stage-specific signal manipulation (Kennedy et al., 2012; Sturgeon et al., 2014; Ng et al., 2016; Luff et al., 2022). These studies paved the way for the generation of blood cells that transcriptomically resemble those found in the aorta-gonad-mesonephros (AGM) embryonic region, where HSCs originate. However these AGM-like hematopoietic stem and progenitor cells failed to fully mimic the functional properties of bona fide HSCs, leading at best to a low-level and transient chimerism in transplanted recipients (Ditadi et al., 2015; Ng et al., 2016; Luff et al., 2022). Recently, the team led by Ng, Stanley, and Elefanty has succeeded in this long-standing endeavor and has successfully “cracked the code” developing a protocol (Fig. 1) that promotes the generation of iPS cell-derived HSCs (iHSCs) capable of long-term engraftment with multilineage capacity in immune-deficient mouse recipients (Ng et al., 2024).

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FIG. 1.

Schematic of the iPS cell differentiation strategy to obtain hematopoietic stem cells. Created with biorender.com.

Based on previous observations, they meticulously optimized a differentiation protocol that is based on the stage-specific manipulation of signaling pathways (in particular TGF-β and WNT) in swirling embryoid bodies and efficiently yields a mesodermal population that is poised to respond to retinoic acid (RA) signaling (Kennedy et al., 2012; Sturgeon et al., 2014; Ng et al., 2016; Luff et al., 2022). Previous studies have shown that RA is a critical signal required for HSC development in vivo and that the addition of retinoids during the specification of the hematopoietic program endows blood progenitors with engraftability, although in short term (Luff et al., 2022).

Building on this, Ng et al. now show that following the WNT-dependent hemogenic mesoderm patterning, the combination of retinoids with the activation of BMP signaling and high concentrations of VEGF is essential for the correct specification of HSCs. In fact, at a later stage, their optimized protocol yields large amounts of hematopoietic cells that also contain HSCs with a robust self-renewal potential. High levels of multilineage human chimerism can be found in recipients at 20 weeks post-transplant, indicating the long-term contribution of iHSCs. BMP4 is expressed in the subaortic mesenchyme surrounding the ventral part of the dorsal aorta where HSC emerges, where it controls the expression of RUNX1, the key transcription factor regulating the emergence of blood cells during development. Regarding RA signaling, how it controls the onset of the HSC program is still unclear. Historically, RA has been shown to affect the expression of Hox genes in vitro and in vivo, but different retinoids treatments have minimal effects on HOXA gene expression in this protocol. Of note, the authors show that a robust HSC specification implies a stage-specific manipulation of VEGF signaling, as later the removal of VEGF facilitated the generation of blood cells. High VEGF levels are typically utilized by the embryo to promote an arterial phenotype. It is worth exploring whether the modulation of Notch signaling, another essential pathway for HSC emergence (Thambyrajah and Bigas, 2022), could also specify HSCs when used in parallel with RA.

Now that the essential sequence of signaling to induce HSCs has been decoded, it is now critical to identify and characterize which of the hematopoietic cells injected are capable of engraftment of iHSCs. To assess multilineage engraftment, the authors injected 0.5–2 million hiPSC-derived hematopoietic cells per mouse. They also noted that engraftment occurred in only 25%–50% of cases, depending on the cell line and protocol, reflecting the rarity of the HSC population generated by their differentiations. This requires a substantial number of CD34+ cells to be injected, with the estimated frequency of iHSCs being less than 1 in 1 million cells.

The authors indicate that human chimerism can be observed in the recipients’ peripheral blood starting around 4 months after injection. While this time might be needed for proper maturation of iHSCs in vivo, it is very likely that this is representative of a limited number of HSCs present in the injected cells. Indeed, the kinetics of engraftment are compatible with those of AGM cells harvested from human embryos at stages where 1–2 bona fide HSCs are present as well as single-cell injection of cord blood-derived HSCs (Ivanovs et al., 2011; Notta et al., 2011). Transplantation studies using neonate recipients can be used to test whether iHSC needs further maturation steps (Arora et al., 2014), in which case it will be important to understand how to replicate these in vitro. The heterogeneity observed by the authors in late-stage cultures might be essential for generating iHSCs, as certain cell types, such as macrophages, could play a critical role in the proper maturation or preservation of HSCs (Mariani et al., 2019; Wattrus et al., 2022). This raises the question of whether it is feasible to generate a population composed entirely of iHSCs.

The article from Ng et al. will serve as a blueprint to answer all these biological questions and will propel the efforts to design personalized HSC-based therapies for a wide range of blood disorders, reducing reliance on donor-derived cells and mitigating issues related to immune compatibility and donor scarcity.