The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cells

Abstract

The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in many developmental processes, including regulation of cell fate and patterning during embryogenesis. Previously, we found that Wnt signaling is required for primitive or yolk sac-derived-erythropoiesis using the murine embryonic stem cell (ESC) system. Here, we examine the effect of Wnt signaling on the formation of early hematopoietic progenitors derived from human ESCs. The first hematopoietic progenitor cells in the human ESC system express the pan-hematopoietic marker CD41 and the erythrocyte marker, glycophorin A or CD235. We have developed a novel serum-free, feeder-free, adherent differentiation system that can efficiently generate large numbers of CD41+CD235+ cells. We demonstrate that this cell population contains progenitors not just for primitive erythroid and megakaryocyte cells but for the myeloid lineage as well and term this population the primitive common myeloid progenitor (CMP). Treatment of mesoderm-specified cells with Wnt3a led to a loss of hematopoietic colony-forming ability while the inhibition of canonical Wnt signaling with DKK1 led to an increase in the number of primitive CMPs. Canonical Wnt signaling also inhibits the expansion and/or survival of primitive erythrocytes and megakaryocytes, but not myeloid cells, derived from this progenitor population. These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development.

Figure 1

Monolayer differentiation of human ESCs generates multipotent hematopoietic progenitors. (A) Representative flow cytometric analyses of ES cells (SSEA4 vs. SSEA3), day 5 hemogenic mesoderm (CD31 vs. KDR) and day 9 hematopoietic progenitors (CD235 vs. CD41) induced in a serum free/feeder free adherent culture system. (B) Flow cytometric analysis of CD235 vs. CD41 expression on the non-adherent population obtained from days 7, 8 and 9 of the adherent differentiation cultures. (C) QRT-PCR gene expression analyses of ES cells and the day 7, 8, and 9 non-adherent cells shown in B. (D) Day 7, 8 or 9 non-adherent cells shown in B were assayed for progenitor colony forming potential and myeloid, mixed (muti-potential) and erythroid colonies were quantified. (E) Cytospins showing May-Grünwald Giemsa staining of myeloid, erythroid and mixed colonies. Scale bars represent 20 μm. Arrows in the myeloid cytospin indicate cells with neutrophil morphology. (F) Non-adherent cells from H9 and H9-GFP ESC lines at days 7, 8 and 9 of differentiation were mixed at a 1:1 ratio and plated in a methylcellulose colony assay. Mixed colonies were scored based upon GFP expression (GFP+: all cells in a colony GFP positive, GFP−: all cells in a colony GFP negative, GFP+/−: mixture of GFP positive and negative cells in a colony). Scale bars represent 500 μm.

Figure 2

Wnt signaling is inhibitory to the generation of CD235+CD41+ early hematopoietic progenitors from human ESCs. (A) Flow cytometric analyses of CD235 vs. CD41 on day 9 non-adherent cells treated with DKK1 (Dkk) or Wnt3a starting on day 4 of differentiation. (B) Cell yields from the entire culture (adherent and non-adherent cells) or non-adherent cells treated as described in A. (C) QRT-PCR gene expression analyses of lineage specific genes in control and DKK1 treated cells. (D) Day 9 non-adherent cells treated as described in A assayed for progenitor potential in methylcellulose and Mega-cult colony assays.

Figure 3

Canonical Wnt signaling inhibits hematopoiesis. (A) Flow cytometric analyses of CD235 vs. CD41 on day 9 non-adherent cells treated on day 4 of differentiation with sFRP1, Wnt5a, GSK3β inhibitor (GSK), and GSK3β inhibitor + Wnt5a starting. (B) Cell yields for the entire culture (adherent and non-adherent) and non-adherent cells treated as described in A. (C) QRT-PCR expression analyses of lineage specific genes in non-adherent cells treated as described in A.

Figure 4

Wnt signaling inhibits the expansion or survival of CD235+CD41+ CMPs. (A) The left flow cytometric profile shows sorted day 9 CD235+CD41+ H9-derived CMPs. The right profiles show these cells cultured for 7 days in hematopoietic cytokines ± DKK1 (Dkk) or Wnt3a and analyzed for markers of megakaryocytes (CD41 vs. CD42), erythrocytes (CD41 vs. CD235), and myeloid cells (CD45 vs. CD18). (B) Cell counts of the cells treated as described in A at days 9, 13 and 16 compared to the untreated (Control) cells. (C) QRT-PCR expression analyses of lineage specific genes in day 4 mesoderm, day 9 sorted CD235+CD41+ cells, and cells treated with DKK1/Wnt3a as described in A. (D) Sorted day 9 CD235+CD41+ cells as described in A plated in methylcellulose and Mega-cult based colony assays with the addition of DKK1 (Dkk) or Wnt3a.

Figure 5

Wnt signaling affects megakaryocyte and erythroid survival/expansion. (A) Flow cytometric profiles of sorted erythroid (CD41−CD235+), megakaryocyte (CD41+CD42+) and myeloid (CD45+CD18+) cells. (B) Sorted cells from A were cultured for 4 days in lineage specific cytokines ± DKK1 (Dkk) or Wnt3a and analyzed by flow cytometry for CD42 vs. CD41, CD235 vs. CD41, and CD18 vs. CD45. (C) Cell counts of two time points for cultures treated as described in B.