Scientists at the Stowers Institute for Medical Research, and the University of Kansas Medical Center, have identified a way to expand blood-forming adult hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB), without differentiating the cells into mature blood cells. If the technique can be translated into a clinical setting, it could make cord blood HSC transplants more readily available to eligible patients with blood cancers, and immune and genetic disorders, for whom a suitable bone marrow donor can’t be found.
Reported in Cell Research, the new approach targets a single protein known as YTHDF2, which impacts on the multiple mRNAs involved in controlling the ability of stem cells to replicate without differentiating. After investigating the concept of YGHDF2 knockdown in a mouse model, the researchers carried out tests in laboratory-grown hUCB samples, which showed that blocking YHDF2 activity boosted eightfold the numbers of functional hUCB HSCs that could be produced ex vivo.
The new approach is also reversible, so the stem cells can differentiate into blood cells after transplantation, and by targeting mRNA, also sidesteps issues with producing any unwanted genetic changes, the researchers suggest. “Our approach of targeting Ythdf2 function using an RNA-based technique also helped avoid more persistent DNA-related changes such as mutations in epigenetic regulators,” explains Zhenrui Li, Ph.D., a predoctoral researcher at the University of Kansas Medical Center. Dr. Li works in the lab of research head Linheg Li, Ph.D., co-leader of the cancer biology program at the University of Kansas Cancer Center and an affiliate professor of pathology and laboratory medicine at the University of Kansas School of Medicine. And because YTHDF2 is present in different types of stem cells, it's possible that the approach could be more widely applied to increase the availability of other types of stem cell therapy for different types of diseases.
The researchers report on the approach in a paper titled, “Suppression of m6A reader Ythdf2 promotes hematopoietic stem cell expansion.”
Only about 30% of patients with blood disorders will have a compatible bone marrow donor in their families. “Life-saving bone marrow transplants have been the common practice for decades, but this doesn't work for everybody,” comments Dr.Li. Transplanting adult stem cells from cord blood is an alternative approach to bone marrow transplantation, which reduces compatibility issues. However, the availability of cord blood and the numbers of stem cells they contain is an issue. One patient may need two cords’ worth of blood, and there aren’t enough cord units for all patients. “If we can expand cord adult stem cells, that could potentially decrease the number of cords needed per treatment,” Dr.Li adds. “That's a huge advantage.”
Previous attempts to trigger cord blood HSC expansion ex vivo have focused primarily on targeting single molecules or pathways. “… it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal,” the authors write. To test the feasibility of this multi-targeting approach Dr. Li’s team focused on YTHDF2, a protein, or “reader,” that recognizes a modification on mRNAs known as m6A. Found on many different mRNAs, m6A regulates the outcome of gene expression by modulating RNA processing, localization, translation, and degradation, the authors explain. In stem cells, m6A plays a role in determining cell fate.
Given this existing knowledge, the authors hypothesized that manipulating YHDF2 could impact on the lifespan of m6A-marked mRNAs, and this could effectively promote the ability of adult HSCs to replicate without differentiation, generating greater numbers for transplantation.
Initial experiments showed that knocking down the YTHDF2 gene in mouse hematopoietic cells increased HSC numbers without changing the types of cells produced, even over the long term. The gene knockout “specifically increases HSC numbers with no bias or defects in either progenitor lineage lines,” the authors write. Using a short hairpin RNA (shRNA) to block YTHDF2 in human umbilical cord HSPCs similarly led to significantly increased levels of ex vivo HSC self-renewal and expansion, resulting in eight times as many functional HSCs when compared with HSC self-renewal in control cells.
Further analyses showed that YTHDF2 recognizes the m6A modification on mRNAs encoding key transcription factors for HSC replication, and speeds the decay of these mRNAs in the stem cells. Effectively, blocking YTHDF2 allowed increased expression of the transcription factors that are critical or self-renewal, “thereby facilitating ex vivo expansion of both phenotypic and functional HSCs without any noticeable lineage bias or leukemic potential.” Encouragingly, there was no evidence that YTHDF2 blockade caused any cell abnormalities or malignancies.
“These data demonstrate that YTHDF2 KD [knockdown] remarkably facilitates the expansion of both phenotypic and long-term functional hUCB HSCs ex vivo,” the authors state. “Our work identifies Ythdf2 as an important regulator of human and mouse HSC self-renewal by coupling the posttranscriptional m6A modification to the degradation of mRNAs encoding key transcription factors for self-renewal.”
YTHDF2 is present in different types of stem cell, so the authors project that their approach could be more broadly applicable. “… we envision that combining our method with previous ones may facilitate the expansion of not only human HSCs, but also other stem cells, offering potential approaches for future stem cell-based therapies.”