A Chemical Approach for Ex Vivo Expansion of Human Hematopoietic Stem Cells
Authors: Nishino Taito , Mikashima Takumi , Yui Makiko , Asai Yasuyuki , Nakauchi Hiromitsu , Iwama Atsushi 
Published: October 2019

Abstract
Abstract 481
Hematopoietic stem cells (HSCs) have been applied to treatment of a wide variety of malignant and non-malignant blood disorders. Because of low risk of graft versus-host disease, use of human cord blood (hCB) as a source of HSCs continues to increase. However, wide application of hCB is limited by the relatively low number of HSCs obtained from a single CB unit. Numerous attempts have been made to expand hCB HSCs in cultures to acquire a sufficient number of transplantable HSCs. Chemical approaches have especially showed promise for ex vivo expansion of HSCs. As an example, Boitano et al. reported that SR1, a chemically synthesized purine derivative, induced hCB HSC expansion by antagonizing the aryl hydrocarbon receptor.
In this study, we initially screened several tens of thousands of small-molecule compounds for the capability to induce STAT5 activation but obtained active compounds showed no positive effect on proliferation of hCB CD34+ hematopoietic stem and progenitor cells (HSPCs). However, a derivative of the STAT5 activating compounds proved to increase the population of CD34+CD38– cells when added to hCB CD34+ cell culture. Thus, we optimized the chemical structure of the compound and identified MISK303 as the most potent compound. We next evaluated the effects of MISK303 on the ex vivo expansion of hCB CD34+ cells in detail; hCB CD34+ cells were cultured in serum-free medium supplemented with MISK303 in addition to rhTPO and rhSCF for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assays. Although the total and CD34+ cell number when cultured with MISK303 was comparable to that cultured with DMSO (vehicle), the cultures with MISK303 exhibited a >2-fold increase in the number of CD34+CD38– cells and contained 1.7-fold more high proliferative potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to those with DMSO. Correspondingly, in the NOD/SCID repopulation assay, hCB CD34+ cells cultured with MISK303 for 7 days displayed up to 2-fold higher levels of engraftment compared to control cultures and uncultured HSPCs. These data suggest that MISK303 promotes the net expansion of hematopoietic stem and progenitor cells. Furthermore, combination of SR1 and MISK303 had additive effects; cell cultures with SR1 and MISK303 showed a >4-fold increase in the number of CD34+CD38– cells. In accordance with the data, MISK303 had no antagonizing activity against the aryl hydrocarbon receptor. We also observed that a chemically-synthesized TPO receptor (c-mpl) agonist could be substituted with rhTPO and provided better expansion of CD34+CD38– cells in combination with SR1 and MISK303.
To investigate the molecular mechanism by which MISK303 increases HSPCs, we first conducted gene expression profiling of CD34+ cells cultured with MK303 by DNA microarray analysis. Treatment of CD34+ cells with MISK303 for 24 hours led to up-regulation of 343-genes (>2-fold) and down-regulation of 112 genes (<0.5-fold). We also profiled effect of MISK303 over 311 protein kinases and found no inhibitory activity. We are now validating the expression of differentially expressed genes identified by DNA microarray analysis.
In conclusion, we have conducted high-throughput screening and identified MK303, a novel chemically synthesized small-molecule compound, which induces the expansion of human HSCs ex vivo. The approach using MISK303 will facilitate the development of novel and efficient technologies for hematopoietic stem cell and gene therapies.

Disclosures:
No relevant conflicts of interest to declare.