LYL1, Class II Basic Helix-Loop-Helix Transcription Factor, Induces the Differentiation of Common Marmoset Embryonic Stem Cells to Hematopoietic Stem Cells

Abstract 2348

Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34⁺ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34⁺ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγ^null) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors.

The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset.

To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34⁺ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1.

Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias.