Optimization of a Clonal Assay for Bipotent Megakaryocyte-Erythroid Progenitors (MEP), and Their Enrichment From Mobilized Peripheral Blood.

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.

Comparison between CD4 and CD8 Lymphocytes as Controls for Methylation-Based Clonal Assay.

In somatic cells from normal female subjects, random inactivation of one of two X chromosomes occurs at an early stage of embryogenesis. Therefore, half of normal females possess an inactivated maternal X chromosome and half possess an inactivated paternal X chromosome. The human androgen receptor gene (HUMARA) contains highly polymorphic CAG trinucleotide repeats and several methyl-sensitive restriction enzyme (me-RE) sites. This polymorphism enables discrimination between paternal and maternal alleles; methylation status can easily be determined using me-RE because unmethylated (activated) sequences are sensitive to me-RE cutting while methylated (inactivated) sequences are resistant. Therefore, in clonal populations, one of the two HUMARA genes should be completely lost. HUMARA-based assays have been applied to evaluate the clonal nature of various hematologic diseases. However, the main drawback of this method is the biased X chromosome inactivation, which is extremely skewed even in normal females. Consequently, HUMARA methylation patterns must be compared with adequate controls and clonal evaluation is impossible when both target and control samples show extremely skewed patterns. In most previous reports, granulocyte lineage clonality is assessed using T-lymphocytes or whole mononuclear cells as controls. To evaluate applicability of CD4 and CD8 cells as controls for clonal analysis, we examined X chromosome inactivation status in 22 healthy heterozygous females. Peripheral CD4+ and CD8+ lymphocytes were separated using the MACS system and DNA was extracted from both samples for PCR-based HUMARA assay. Although the 2 HUMARA peaks were almost identical in the HpaII pre-digested samples, there was skewing in some samples from elderly subjects after HpaII digestion: only 3 females in the CD4 group showed biased X inactivation, while 7 females in the CD8 group showed biased inactivation. These results indicate that CD8+ lymphocytes tend to more frequently show age-dependent skewing when compared with CD4+ lymphocytes. Because it has been reported that the frequency of peripheral naive T lymphocytes is reduced in advanced age, we investigated the correlation between CD28 expression and age in 25 healthy controls (median age of 67), as the majority of CD28-positive cells were naive T cells. The proportion of the naive T cell population that was composed of CD8+ lymphocytes decreased markedly with age when compared with that of CD4+ cells. Subsequently, we investigated age-related T cell receptor repertoire alterations. To evaluate the complexity of the TCR repertoire, the TCR Vβ repertoires of CD4+ and CD8+ lymphocytes were assessed by CDR3 size distribution analysis. The mean complexity score for control CD8+ lymphocytes (72.0) was significantly lower than that for CD4+ ymphocytes (126.5, P=0.011). In conclusion, frequent skewing of X chromosome inactivation was paralleled by severe reductions in naive T lymphocytes and T cell repertoire in CD8+ lymphocytes. Therefore, CD4+ lymphocytes might be better suited as controls for methylation-based clonal assay than CD8+ lymphocytes.

Establishment of Clonal Colony-Forming Assay System for Pancreatic Stem/Progenitor Cells

Pluripotent stem cells found in a number of organs are usually in small cell populations. However, under adaptive stimulation, they enter the stage of growth and differentiation to compensate for the loss of differentiated cells. To analyze stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are strongly required. In this study, we established a colony-forming assay system for pancreatic stem/progenitor cells in vitro. In this culture condition, they received signals for growth and differentiation, and formed clonal colonies including pancreatic endocrine-lineage cells, such as α and β cells. By combining this culture system with flow cytometric cell sorting, pancreatic stem/progenitor cells will be enriched, and their potential can be analyzed precisely in single cell-based experiments.