Generation of Megakaryocytes and Platelets from Valproic Acid Expanded Human Cord Blood CD34+ Cells in Culture

Currently in the US alone there are more than 2 million units of platelets transfused each year. The transfusion of platelets can be crucial life saving measures for patients undergoing treatment for leukemia and other cancers. However, volunteer donors are the only source of platelets for transfusions. The objectives of our studies are to further refine our multi-step culture strategies to expand human umbilical cord blood (CB) derived megakaryocyte (MK) progenitors, and augment functional MK/platelet yields. We used serum-free culture to expand stem/progenitor cells over 5 to 7 days from CB and growth factor mobilized peripheral blood (mPB) CD34⁺ cells using valproic acid (VPA), a histone deacetylase inhibitor (HDAC). VPA-expanded CB cells were assayed for MK progenitors by flow cytometry and CFU-MK assays. Compared to mPB, CB CD34⁺ cells yielded relatively more CD34⁺CD90⁺ cells (>1.6 fold), yet the absolute numbers of CD34⁺CD41a⁺ MK progenitors were comparable (CB 7.18 ± 1.19 x 10⁵/well vs. mPB 7.55 ± 1.14 x 10⁵/well). The CFU-MK colony yield from mPB was relatively lower than CB (CB 32.31 ± 9.57 x 10³/well vs. mPB 11.3 ± 5.74 x 10³/well, not significant). Extending the CB cultures up to 7 days resulted in notably greater fold increase of CD34⁺CD90⁺ primitive stem/progenitor cells from prior to expansion (D5 VPA 75.77 ± 14.83 vs. D7 VPA 146.26 ± 28.52-fold increase of CD34+CD90+ cells; p = 0.07, n = 4) as well as increase in CD34⁺CD41a⁺ MKs (D5 VPA 1.02 ± 0.24 X 10⁵/well, n = 8, vs. D7 VPA 2.29 ± 0.85 X 10⁵/well, n= 6, p = 0.04) and slightly higher number of CFU-MK colonies. We then estimated CD41a⁺ MK cell yield per initial single CD34⁺ CB cell plated to initiate expansion culture. VPA-expanded CD34⁺ cells were further cultured in the presence of various small molecules including stem reginin 1 (SR1), UM171, VPA, and nicotinamide. The yield of CD41a⁺ MKs from a single CD34⁺ cell plated increased with time. Having UM171 or VPA in extended cultures is favorable to generate high yield CD41+ MKs. Interestingly, a single CD34⁺ cell following VPA-expansion yielded up to 2,060.32 ± 631.48 (mean ± SE, n= 6) CD41a⁺ MKs after 17 days of culture. If we plated every single cell from a CB unit containing 5 million CD34⁺ cells in VPA expansion culture and continued the cultures for 17 days, 10.3 billion CD41a⁺ cells (10.3 x 10⁹) could be generated. If we are capable of producing about 50 platelets per MK plated using optimized culture conditions, this would yield almost 2 units of transfusible platelets (5.15 x 10¹¹) for patients. Using cultures of CB CD34⁺ cells, the relative expression levels of 12 genes were assessed by real-time qRT-PCR. Genes tested were classified according to the relative abundance of transcripts for the erythroid ( EPOR and KLF1 ) and MK ( AML1 , FLI1 , GABPA , and MPL ) lineages, as well as genes common to both lineages ( GATA1 , NFE2 , TAL1 , GATA2 , MYB , and LMO2 ). Interestingly in MK inductive culture, VPA treated D14 samples displayed relatively lower transcript levels of erythroid genes including EPOR and KLF1 and higher expression of MK specific gene Fli1. After further differentiation at day 20 the expression of MK specific gene FLI1 achieved the highest level of expression (D0 to D7 1.84 ± 0.3; D0 to D20 3.52 ± 0.79-fold increase) and erythroid gene KLF1 had the lowest level of expression (D0 to D7 12.15 ± 1.72; D0 to D20 5.76 ± 3.77-fold increase) relative to pre-culture samples. Taken together, MK specific gene transcript levels increased while erythroid specific gene expression decreased following extended culture of CD34+ cells. These results suggest that VPA expanded CB cells maintain MK lineage commitment during extended culture. The MKs generated in culture appear to be relatively immature and only 21.44 ± 2.99 % (n = 4) of CD41a+ MKs co-expressed CD42b. However, in optimal MK condition the VPA expanded CB cells were capable of generating mature MKs (CD41a+/CD42b+) leading up to 3 to 7 times more CD41a+CD42b+ MKs in comparison to unexpanded CB CD34+ cells. Platelets generated per CD41a+CD42b+ MKs from VPA-expanded CD34+ cells were also 2 to 4 times greater than unexpanded CD34+ cells. Our current MK generation strategies from VPA-expanded CB cells, once further scaled up and optimized for MK maturation/platelet generation, has the potential to generate about two units of platelets for transfusion.