Screening of Hematopoietic Small Molecules Facilitates the Establishment of a Chemically Defined Hsc Expansion Medium With Enhanced Performance

Hematopoietic stem cells (HSC), known for their ability to multipotent and self-renew, are often used in HSC transplantation for the treatment of hematological diseases and malignancies. Umbilical cord blood (UCB) and mobilized peripheral blood (mPB), which are alternatives to bone marrow (BM) for HSC transplantation, have reduced HSC. To address this restriction, the ex vivo expansion of HSCs is a highly promising therapeutic approach rather than the use of a double-cord blood unit that induces delays in hematopoietic recovery. We have previously shown that knockout of HSC quiescence genes could increase the HSC pool in vivo. Thus, we thought that targeting HSC quiescence regulators using small molecules could be used for ex vivo expansion of both mPB and UCB-HSC. The goal was to identify novel hematopoietic small molecules (HSMs) and their combinations, and to enhance performance of human HSC expansion medium. We identified and analyzed 35 possible HSMs targeting HSC quiescence factors. We assessed their impact on human HSPC activity, including expansion, quiescence, multilineage capacity, cycling capability and metabolism. We have also investigated their cytotoxic and genotoxic effects on human HSPCs. On the other hand, a transplantation study was performed on immunocompromised mice for the evaluation of the repopulation and engraftment capacities of the expanded cells. We observed that BML-260 and TAME molecules robustly increased both the mPB and UCB-HSPC content and activated the re-entry of HSCs into the cell cycle. Colony Forming Unit (CFU) assay confirmed their improved multilineage capacity. BML-260 proved safer for the viability of expanded cells based on cytotoxicity and genotoxicity assays. In addition, gene expression analysis showed that BML-260 and TAME molecules contributed to HSC expansion by modulating cell cycle kinetics, including p27, Skp2 and Cdh1. In conjunction with these in vitro results, we have observed that BML-260-expanded HSCs had a strong hematopoietic reconstitution capacity. After the determination of the most effective molecule as BML-260, a comparative study of chemically defined media, including various supplements, was analyzed in addition to the BML-260 molecule. These results from in vitro and xenotransplantation experiments have shown that BML-260 molecules can be used therapeutically for human HSC expansion and regulation of HSC activity. In addition, the medium composition found may be a novel platform for human HSC expansion to used in clinical trials.

Human Erythroblasts with c-Kit Activating Mutations Have Reduced Cell Culture Costs and Remain Capable of Terminal Maturation and Enucleation

There is a constant need for red blood cells for transfusion therapy in the treatment of anemias and acute injury. As all blood products for transfusion come from donors, there are concerns over shortages and safety. Furthermore, many patients with transfusion-dependent anemias risk alloiumminization. The in vitro production of red blood cells would address these problems, especially as they can be genetically engineered to prevent alloimmunization. Numerous erythroid culture systems now exist for the in vitro production of red blood cells. Hematopoietic stem and progenitor cells (HSPCs) obtained from umbilical cord or peripheral blood can be differentiated into erythrocytes, however, they are limited in expansion. While umbilical cord HSPCs have greater expandability than peripheral blood, the resulting erythrocytes contain fetal globins. Pluripotent stem cells can also be used as a starting source, however only a small percentage of the cells can be differentiated into erythroblasts which also suffer from low enucleation rates. Presently, the cost of in vitro production of a unit of red cells is greater than an order of magnitude higher than obtaining it from a donor largely due to the medium and cytokine costs (Timmins & Nielsen, Trends Biotechnol, 2009). A relatively new approach of immortalizing early erythroblasts allowing unlimited expansion as well as terminal maturation and enucleation shows great therapeutic promise (Kurita et al., PLoS One, 2013; Huang et al., Mol Ther, 2014; Trakarnsanga et al., Nat Commun, 2017). However, these immortalized erythroblasts are still reliant on two costly cytokines: stem cell factor (SCF) and erythropoietin (Epo). Mutations in exon 17 of the receptor tyrosine kinase gene KIT are frequently seen in acute myeloid leukemias, gastrointestinal stromal tumors, and mast cells leading to mastocytosis. These mutations cause the c-Kit protein to spontaneously activate and transduce signal in the absence of SCF (Kit-ligand). To generate an SCF-independent HUDEP-2 cell line (Kurita et al., PLoS One, 2013), we used CRISPR/Cas9 to introduce missense and frameshifting mutations within the vicinity of Asp816 in exon 17 of the KIT gene. The resulting monoclonal cell lines were selected for by removing SCF from the expansion medium and were subsequently named KIT-CAT (KIT with Constitutively Activating Transformation). To better understand what KIT mutations allowed or impaired terminal maturation, monoclonal cell lines were genotyped by Sanger sequencing. Three cell lines with unique genotypes were chosen for further analysis. All three KIT-CAT lines had a shorter doubling time compared to HUDEP-2 cells (16.7 vs 18.9 hrs, p=0.020) and were no longer dependent on SCF or Epo. However, two of the three KIT-CAT lines showed more robust proliferation with Epo in the expansion medium. The addition of SCF to the medium caused no increase in c-Kit activation by Western blotting for phosphorylation at Tyr703. Furthermore, the low molecular weight and immature form of c-Kit is also phosphorylated in KIT-CAT cells, but not HUDEP-2 cells, indicating c-Kit activation occurs before trafficking to the cell membrane where SCF would bind (Tabone-Eglinger et al., Clin Cancer Res, 2008). Key features of erythroblast maturation are the decrease in cell and nuclear size which can be measured using imaging flow cytometry (McGrath et al., Methods, 2017). While in expansion phase, all 3 cell lines were larger in cell and nuclear area compared to the parental HUDEP-2 line. By day 6 of maturation, all three cell lines had statistically significant decreases in cell and nuclear size indicating maturation. By day 13 of culture, Wright-Giemsa staining showed that the majority of the cells were orthochromatic erythroblasts or enucleate reticulocytes. Reducing cell culture costs is needed for in vitro manufacturing of red blood cells to be economically feasible. These results show that a c-Kit activating mutations in human erythroblasts removes the cost of SCF and reduces the cost of Epo while still allowing for terminal maturation and enucleation. Disclosures No relevant conflicts of interest to declare.

Characteristics of Human Natal Stem Cells Cultured in Allogeneic Medium

Recently, human natal dental pulp stem cells (hNDP-SCs) have been characterized in vitro and it has been shown that they satisfy criteria defining human mesenchymal stromal cells (MSCs), as proposed by the International Society for Cellular Therapy. However, these results were reached in the presence of xenogeneic expansion medium, which has the potential to alter the cells’ functional capacity. To determine the validity of the previously reported hNDP-SCs characteristics for human cell therapy, we have cultured hNDP-SCs in allogeneic expansion medium. Two hNDP-SC lineages were isolated from vital natal teeth, donated by a healthy newborn female and cultured in 2% platelet rich plasma (PRP). Analysis of the phenotypic expressions, proliferation rates, viability, telomerase length and in vitro adipogenic, osteogenic and chondrogenic differentiation potentials of two hNDP-SCs lineages (Zn001 and Zn002) were performed. Both lineages displayed similar morphology, proliferation rates, adipogenic, chondrogenic and osteogenic differentiation potential. Telomere shortening by 41.0% and 13.49% occurred from 3rd till 14th passage for lineages Zn001 and Zn002 respectively. Viability of both lineages was higher than 90%. Flow cytometry demonstrated that both lineages were positive to the majority of tested markers, including markers, which were negatively, expressed when hNDP-SCs were cultured previously in xenogeneic medium. Using immune-cytochemistry the cells were shown to express beta III-tubulin, nestin, neurofilaments and Nanog. PRP used as allogeneic medium is suitable for cultivation of hNDP-SCs.