Endocytosis as a critical regulator of hematopoietic stem cell fate —implications for hematopoietic stem cell and gene therapy

Abstract

Hematopoietic stem cells (HSCs) have emerged as one of the most therapeutically significant adult stem cells, paving way for a range of novel curative regimens over decades. HSCs are transplanted, either directly or post restorative genetic engineering in order to repopulate a healthy hematopoietic homeostasis in patients with disorders affecting the blood and immune cells. Despite being an extensively studied system, the maintenance and expansion of functional HSCs ex vivo remains a major bottleneck. The challenge primarily stems from difficulties in reproducing HSC self-renewal divisions and gradual depletion of stemness characters, in vitro. Refining the in vitro culture can be particularly beneficial in the case of cord blood HSCs (CB-HSCs), as inadequate numbers in a single umbilical cord limits its therapeutic potential. In recent years, molecular dissection of HSC stemness has significantly improved in vitro hematopoietic stem and progenitor cells (HSPCs) culture. Despite such significant progress, lacunae exist in fully understanding all the underlying mechanisms and their interplay active in bona fide HSCs, and how it transforms when cells proliferate in culture. A new groundbreaking study titled “MYCT1 controls environmental sensing in human haematopoietic stem cells”, published in Nature in June 2024, sheds light on this complex field. Through a series of experiments, including knock-down, overexpression, single-cell RNA sequencing, and transplantation, the study identifies a previously unknown role of the MYC target 1 (MYCT1) protein in HSC maintenance. This protein acts as a crucial regulator of human HSCs, with high expression in primitive HSCs and subsequently downregulated during ex vivo culture. The study reveals that MYCT1 plays a vital role in moderating endocytosis and environmental sensing in HSCs, processes thereby essential for maintaining HSC stemness and function. This commentary will discuss the implications of the new findings for cord blood expansion in cell therapies and HSPC culture for gene therapy applications, providing valuable insights for the field of hematopoietic regenerative medicine.

Background

Researchers have long sought to expand hematopoietic stem cells (HSCs) from cord blood to quantities sufficient for transplantation, which could potentially save patients lacking compatible human leukocyte antigens (HLA)-matched donors [1, 2]. However, this goal remains elusive despite advanced high-throughput screens identifying factors that support ex vivo culture and expansion of HSPCs [3, 4]. Most screens have relied on immunophenotypic markers of HSCs as readouts, which only partially correlate with HSC engraftment potential. New markers like Endothelial protein C receptor (EPCR), Integrin-α3 (ITGA3) and hepatic leukemia factor (HLF) have emerged through the years. These markers enhance the correlation between marker expression, the HSC transcriptome and engraftment potential, aiding in the identification of functional long-term HSCs (LT-HSCs) [5, 6]. Yet even these markers fail to fully capture functional HSCs generated ex vivo. The identification of intranuclear factors such as Mixed-Lineage Leukemia Translocated to Chromosome 3 Protein (MLLT3) which regulates H3K79me2 and maintains an open chromatin state of key HSC regulatory genes, including MECOM, HLF, and Musashi-2 (MSI2), has improved our understanding of HSC stemness and self-renewal [3]. This also signifies the need to elucidate such regulatory programs, which could potentially lead to targeting synergistic pathways that support robust expansion of functional HSCs.

In this pursuit, Aguadé-Gorgorió et al., conducted RNA sequencing analysis to examine the expression of HSC factors in haematopoietic tissues across development and examined the same factors when HSCs were subjected to in vitro culture [7]. This RNA-seq-based strategy marks a shift from relying solely on surface markers to incorporating transcriptomic data for a more comprehensive understanding of HSC biology. By pinpointing MYCT1 as a key player in HSC regulation, this study opens new avenues for potentially improving ex vivo HSC expansion methods.

Main text

Significance of MYCT1 and endocytosis in HSC regulation

Hematopoietic stem cell fate regulation has been widely studied from the angle of transcriptional and epigenetic programs co-ordinating self-renewal and differentiation. Such studies have resulted in the development of aryl hydrocarbon receptor antagonists and compounds preserving epigenetic marks for expanding HSCs during ex vivo culture, and, in turn, CB-HSCs expanded ex vivo with these candidates, have been promising in early clinical studies [1, 8]. The study by Aguadé-Gorgorió et al., presents “endocytosis” as another crucial program regulating HSC fate [7]. Low endocytosis-mediated regulated environmental sensing is key for HSC maintenance, and it suggests that HSCs require stricter control of environmental sensing mechanisms compared to progenitors and differentiated cells. Hence, a tightly controlled endocytosis axis may be crucial for maintaining the unique functional properties of HSCs. The results uncover another layer of complexity in signal sensing programmes in HSCs, with an earlier study reporting an interesting phenomenon of controlled lysosomal activity in phenotypic LT-HSCs, which further alters the cell fate determination trajectories [9]. Moreover, MYCT1 is highly expressed in MLLT3 and HLF expressing fractions, indicating that MYCT1 expression corroborates with other stemness signatures, and can potentially be a novel HSC hallmark. This insight could lead to new strategies for identifying and isolating high-quality HSCs based on their endocytosis rates or MYCT1 expression levels, in addition, or as an alternative to current LT-HSC immunophenotypic markers; CD34 + CD133 + CD90 + CD49f + cells, potentially improving the efficacy of HSC transplantations and gene therapies.

Implications for ex vivo HSC expansion

Stromal cells or small-molecule compounds like Stemreginin1 and UM171 have been shown to support the in vitro culture of HSCs [1, 10], but the new study points that these systems fail to maintain the expression of MYCT1 or the endocytosis rates. This clearly underlines that the existing ex vivo expansion systems are insufficient to fully preserve the functional integrity of HSCs. It would be interesting to screen the expansion supporting small molecule compounds incorporating MYCT1 expression as a readout. Also, current methods using high doses of cytokines or small molecule compounds may inadvertently increase endocytosis rates, potentially compromising HSC quality. It would also be interesting to test whether, the recently proposed, chemically defined cytokine-free HSC culture systems can sustain the MYCT1 expression [11]. The observation that restoring MYCT1 expression can suppress excessive culture-induced endocytosis in HSPCs can be further explored as a new approach to maintaining HSC function during ex vivo expansion.

This research raises important questions about current HSC culture methods:

Should we re-evaluate the use of high-dose cytokines in HSC culture?

Can we identify upstream regulators of MYCT1 expression to develop more effective expansion protocols?

Should we supplement endocytosis blockers to HSC culture media?

Impact on HSPC gene therapy

The implications of this study extend to the rapidly advancing field of HSPC gene therapy, which encompasses gene editing, retroviral (lentivirus) or adenoviral gene addition techniques. With the recent commercial approval of HSPC gene editing therapies [12], understanding the factors that maintain HSC quality during manipulation becomes crucial. Homology-directed repair (HDR) gene editing of HSPCs or lentiviral mediated manipulation takes at least 2 days of ex vivo culture. It would be interesting to look at the effect of gene-manipulation stress on the MYCT1 expression. MYCT1 could serve as a valuable quality control indicator for gene-edited HSPCs, potentially improving the outcomes of these therapies.

Moreover, recent findings suggest that shorter culture periods or even culture-free gene editing methods may be beneficial for preserving HSC function [13, 14]. This could lead to the development of more efficient and effective protocols for genetic modification of HSCs, enhancing the potential of personalized cell therapies (Fig. 1).

Fig. 1

Schematic illustration of MYCT1-associated stemness and metabolic programs in ex vivo expanded HSPCs. Potential therapeutic benefits of sustained MYCT1 expression for hematopoietic stem cell and gene therapy are also indicated. Image created with Biorender.com

Future directions and conclusion

This research opens up several avenues for future investigation:

Elucidating the specific endocytic pathways through which MYCT1 exerts its effects on HSC function.

Developing methods to sustain MYCT1 expression during ex vivo culture without relying on viral vectors.

Exploring the potential of MYCT1 and endocytosis rates as markers for HSC quality in clinical applications.

In conclusion, the discovery of MYCT1’s role in regulating HSC fate through the control of endocytosis represents a significant advancement in hematopoietic stem cell biology. By highlighting the importance of controlled environmental sensing in quiescent HSC maintenance, this study not only deepens our understanding of fundamental haematopoietic stem cell biology but also offers new strategies for improving HSC expansion protocols and cell therapies. As research in this area progresses, it has the potential to revolutionize HSC-based regenerative medicine strategies to cure diseases.