Optimization of a Clonal Assay for Bipotent Megakaryocyte-Erythroid Progenitors (MEP), and Their Enrichment From Mobilized Peripheral Blood.
Authors: Sanada Chad D , Zhang Ping-Xia , Zerafati Gazelle , Krause Diane S 
Published: October 2019

Abstract
Abstract 2310
Megakaryocyte/Erythroid Progenitor (MEP) cells are bipotent hematopoietic progenitors that undergo a fate decision to commit to either Megakaryocyte (Mk) or Erythroid (E) lineage. How this biphenotypic fate decision is made is not clearly understood. In order to elucidate the epigenetic changes underlying the MEP fate decision in human cells, one must first have a robust approach for purifying the starting population of MEP. Published enrichment strategies for human MEP are suboptimal as they also enrich committed erythroid progenitors, and assays aimed at identifying MEP are limited as they do not demonstrate single cells differentiating down both the Mk and E lineages. Thus, refined strategies for purifying and assaying MEP will greatly facilitate future studies of the MEP fate decision. Our aim was to develop an optimized assay for identifying MEP and to then directly compare different MEP purification strategies. We developed a clonal assay for biphenotypic cells using collagen based, semi-solid media containing cytokines that promote Mk and E differentiation (Tpo, Epo, SCF). In order to visualize Mk and E colony formation, we optimized dual immunohistochemistry for CD41a (Mk specific) and GlyA (E specific) with which we could distinguish colonies that were purely erythroid cells, purely megakaryocytic, or a combination of both E and Mk. Using this assay, we compared the efficiency and purity of three enrichment strategies for human MEP that were published by the laboratories of Tor Olofsson, John Dick, and Irving Weissman. The three “MEP” populations were all FACS sorted from mobilized peripheral blood (mPB) cells starting with Lin-CD34+CD38+CD45RA- cells. They were then stained separately for one the following antigenic phenotypes: TpoR+ (8.5–13% of parent population, Olofsson); Flt3− (12.5–14.5% of parent, Dick); or IL-3Ra− (4–13% of parent, Weissman). Sorted cells were plated in both methylcellulose (MeCe) (125 cells/dish) and collagen-based assays (250 cells/2 dishes) and cultured for 13–15 days before scoring colonies. For MeCe assays, colonies were scored as Granulocyte/Erythroid/Monocyte/Megakaryocyte (GEMM), Erythroid (E), or Granulocyte/Monocyte (GM), which had granulocytes and/or monocytes. For collagen-based assays, colonies were scored as megakaryocyte/erythroid (Mk/E), erythroid only (E), or megakaryocyte only (Mk). The results (see table) showed that the three sorting strategies gave different results. In MeCe, the total plating efficiency was 25%, 50% and 38% for the 3 sorting strategies, respectively. With regard to Mk/E biphenotypic colonies, the TpoR+ population had the highest percentage of Mk/E (32.0 ± 1.9% of total colonies), while the Flt3− (19.6 ± 2.9%) and IL-3Ra− (20.7 ± 6.2%) populations showed lower levels. On a per cell basis, however, the cloning efficiency of Mk/E colonies was similar among all three with the highest in Flt3− cells (11.2 ± 3.5%) followed by TpoR+ cells (10.8 ± 0.0%) and IL-3Ra− cells (9.7 ± 2.3%). The difference in percentage of Mk/E colonies between the 3 methods may be due in part to the presence of varying amounts of ‘contaminating’ GM colonies in MeCe. TpoR+ cells gave rise to the highest amount of GM colonies (12% GM, n=1) on a per cell basis followed by IL-3Ra− cells (8.4 ± 0.6% GM) and Flt3− cells (4.0 ± 0.0%). Taken together, the results demonstrate that all 3 MEP purification strategies enrich for Mk/E colony forming potential from mPB samples. However, none of the methods is adequate to achieve a high purity (>50%) of Mk/E colony forming cells, and all are more enriched for E only and/or Mk only colony forming potential. In order to address this problem, we are now combining the most favorable MEP markers into a single stain for assessment of colony forming efficiency and purity of MEP. Using this refined approach to sorting MEPs will allow detailed epigenetic studies to be performed that will shed new insight into the MEP fate decision.
Cell Surface Phenotype TpoR+ Flt3- IL-3Ra- Total cloning freq (collagen) 34 ± 2% 56 ± 9% 48 ± 5% E % of colonies (collagen) 22 ± 7% 55 ± 18% 66 ± 14% Mk % of colonies (collagen) 46 ± 5% 25.7 ± 15% 13 ± 8% Mk/E % of colonies (collagen) 32 ± 1.9% 20 ± 2.9% 21 ± 6% Total cloning freq (MeCe) 25% 50 ± 0.6% 38 ± 3% GM % of colonies (MeCe) 48% 8 ± 0.1% 22 ± 3%

Disclosures:
No relevant conflicts of interest to declare.