High Levels of CD44 Expression Identify Hematopoietic Cells Capable of Generating Great Numbers of Erythroid Cells Under HEMA Conditions
Authors: Migliaccio Anna Rita F , Tirelli Valentina , Masiello Francesca , Sanchez Massimo , Whitsett Carolyn , Migliaccio Giovanni 
Published: October 2019

Abstract
Abstract 3349
Both mononuclear (MNC) and CD34pos cells from adult blood (AB) generate great numbers of erythroid cells (EBs) in cultures when stimulated with dexamethasone, estradiol, SCF, IL-3 and EPO (Human Erythroid Massive Amplification, HEMA). The total number of erythroid cells generated by CD34pos cells is, however, on average 1-log lower than that generated by MNC. Loss of erythroid progenitor cells during the CD34 selection procedure and/or existence of circulating CD34neg erythroid progenitors have been considered as possible explanations for this observation. To clarify the phenotype of the circulating hematopoietic cells that generate EBs under HEMA conditions, we compared progenitor cell activity (CFC, i.e. generation of BFU-E and CFU-GM derived colonies in semisolid media,) and erythroid precursor activity (EPC) (i.e. fold increase, FI, of EBs at day 15 in HEMA) of cell populations prospectively isolated from AB MNC by cell sorting according to expression of CD34, CD44 (the α integrin subunit which represents an early erythroid marker in mouse, Chen et al, PNAS 2009, 106:17413-17418), CD36 (the thrombospondin receptor whose expression is activated when hematopoietic progenitors are exposed to EPO) and CD42a (the α subunit of von Willebrand factor, a megakaryocyte marker as negative control). The results are summarized in Table I.
Table I. Populations present in AB MNC with the potential to generate erythroid cells under HEMA conditions     CD44 high high high neg     CD34 pos neg neg neg     CD36 neg neg low high     CD235a neg neg neg neg     CD42a neg neg neg pos Frequency 0.1% 78% 0.84% 0.56% CFC 160/500 plated cells (BFU-E:CFU-GM = 1:1) 17/10,000 cells (all BFU-E) – – EPC FI = 12,000 Day 15 FI = 15 Day 15 FI = 1,250 – Other Megakaryocyte precursors (?)
At least three cell populations were recognized to exert EPC activity: CD34pos/CD44high cells, which grew in HEMA culture (FI = 12,000) and contained CFC (160 CFC/500 plated cells); CD34negCD44pos cells (78%) which generated EBs in HEMA (FI = 15) and contained CFC (16.5±0.5 BFU-E/10,000 plated cells) and CD34negCD44posCD36low cells which generated EBs in HEMA (FI = 1250) but did not contain CFC. An additional population of CD36high cells which did not express CD14 and were therefore not monocytes, did not express CD44 but expressed CD42a. These cells did not generate EBs in HEMA or CFC in semisolid cultures and were likely megakaryocyte precursors. Therefore, the three populations capable to generate erythroid cells in HEMA expressed CD44 at high levels.
These results suggest a pattern of antigenic activation during erythroid commitment in which CD44 is already expressed by CD34pos cells. Its expression is retained at early stages of erythroid commitment until low levels of CD36 expression appear and is lost when cells upregulate CD36 expression to acquire MK markers (CD42a). To test this model, we analysed the changes in antigen expression profiles of cells generated by MNC during 13 days of HEMA culture. The results are summarized in Table II.

Table II. Dynamic changes in antigen expression during the culture of MNC in HEMA (in percent of total cells) CD44 high high high high medium low CD34 pos neg neg neg neg CD36 neg low low high high low CD42a neg neg neg neg neg neg CD235a neg neg neg neg pos pos Day 0 0.1 – 0.84 – – – Day 1 0.1 ± 0.4 – – – Day 2 0.2 0.1 0.6 – – – Day 3 0.7 0.3 0.9 – – – Day 6 – – 1.1 – 0.2 – Day 8 – – – 19 39 – Day 10 – – – 19 63 – Day 13 – – – 13 77 3.6 Results are expressed as percent of the total population present in culture. CD44posCD34negCD36neg cells were not analysed in these experiments. Absorbance fluorescence intensity for CD44: High= >8.000; medium=1000–2000; low=500–800
CD34pos cells increased from 0.1 to 0.7% at day 3 of culture and became undetectable by day 6. CD34negCD36low cells increased in frequency until day 6 when they represented 1.1% of the total cell population. A CD34posCD36low population, not detectable in MNC, appeared in culture at day 1 and represented 0.3% of total cells at day 3. These cells may represent the progeny of CD34pos cells that have acquired CD36 expression. All these cells retained high levels of CD44 expression. Another population not detectable among MNC was represented by CD34negCD44highCD36high CD42aneg cells. These cells appeared at day 6 and became 19% of the total cells by day 8. Prospective isolation coupled with functional studies identified that these cells have EPC activity as demonstrated by their ability to generate additional EBs in HEMA. Cells expressing the erythroid marker CD235a (glycophorin A) began to be detectable by day 6 (0.2%) and reached 77% of the total population by day 13. These cells expressed medium levels of CD44 and when prospectively isolated were unable to generate additional EBs in HEMA. By day 13, few cells (3.6%) with the phenotype of mature EBs (CD36lowCD235ahigh) were detectable. These cells expressed low levels of CD44.
Overall, these data indicate that high levels of CD44 expression mark in vivo and ex vivo generated cells with EPC activity.

Disclosures:
No relevant conflicts of interest to declare.