Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model

Abstract

Until now, ex vivo generation of CD34(+) hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14(+) monocyte-derived macrophages to generate CD34(+) cells from hESCs in vitro. Human ESC-derived CD34(+) cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow-derived CD34(+) cells. Finally, transplantation of purified hESC-derived CD34(+) cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This in vivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34(+) cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.

Figure 1. Combination of MSCs and macrophages simulates bone marrow microenvironment

(A) The level of gene expression was not statistically different between MSC-educated macrophages (MEM) and bone marrow macrophages (BM MQ) in most of the genes tested except TNFRSF13B (p value <0.05), suggesting similarity between two population. (B) Principal component analysis on MEM, BM MQ samples with peripheral blood monocyte derived macrophages (MQ, n=3) shows clustering of MEM with BM MQ, away from MQ. (C) MSCs and macrophages were able to support proliferation of CD34+ cells and the combination of MSCs and macrophages increased proliferation of CD34+ cells more than either cell type alone.

Figure 1. Combination of MSCs and macrophages simulates bone marrow microenvironment

(A) The level of gene expression was not statistically different between MSC-educated macrophages (MEM) and bone marrow macrophages (BM MQ) in most of the genes tested except TNFRSF13B (p value <0.05), suggesting similarity between two population. (B) Principal component analysis on MEM, BM MQ samples with peripheral blood monocyte derived macrophages (MQ, n=3) shows clustering of MEM with BM MQ, away from MQ. (C) MSCs and macrophages were able to support proliferation of CD34+ cells and the combination of MSCs and macrophages increased proliferation of CD34+ cells more than either cell type alone.

Figure 2. CD34+ cells generated from hESC-MSC-Macrophage coculture

The gating strategy used for cocultured cells (A) and representative flow cytometry data (B). Among the live cell gating, about 20% of cells were CD14 positive macrophages while about 10% of cells stained positive for CD34 positive cells. (C) The percentage of CD34+ cells present vs. days in culture from one representative culture. The level of CD34 positive cells reached maximum around day 11 and subsequent culture exhibited decreased level of CD34 positive cells. (D) The cell surface marker expression of CD34+ cells differentiated from hESCs. CD26, CD117, CD133/1, CD309 and anti HPC antigen are expressed in a statistically significant way.

Figure 2. CD34+ cells generated from hESC-MSC-Macrophage coculture

The gating strategy used for cocultured cells (A) and representative flow cytometry data (B). Among the live cell gating, about 20% of cells were CD14 positive macrophages while about 10% of cells stained positive for CD34 positive cells. (C) The percentage of CD34+ cells present vs. days in culture from one representative culture. The level of CD34 positive cells reached maximum around day 11 and subsequent culture exhibited decreased level of CD34 positive cells. (D) The cell surface marker expression of CD34+ cells differentiated from hESCs. CD26, CD117, CD133/1, CD309 and anti HPC antigen are expressed in a statistically significant way.

Figure 3. Comparison of gene expression of CD34+ cells from different origin

The expression of 13 hematopoietic genes in were analyzed and then normalized to the average for 3 housekeeping genes (18S rRNA, GAPDH and b-Actin) for each sample. (A) BCL2A1, CD34, EGR1, GATA2 and RUNX1 were up-regulated compared to hESCs in both hESC-derived CD34+ cells (hESC CD34+, n=6) and bone marrow CD34+ (BM CD34+, n=6) with statistical significance (t-test, p value < 0.05). GATA3 and ID1 were up-regulated in hESC CD34+ while FLT3, LMO2 and MCL1 were up-regulated in BM CD34 compared to hESCs. (B) Principal component analysis using gene expression data from hESC (n=4), hESC-derived CD34+ (n=5), BM CD34+ (n=2) and mobilized peripheral blood CD34+ cells (PB CD34+, n=3). hESC CD34+ (Component 1 average value = 20.4) showed shift toward BM CD34+ and PB CD34+ (Combined component 1 average value = 7.9) away from hESCs (Component 1 average value = 31.2) along the principal component 1 which accounted for 72.8% of total variation within the data set.

Figure 4. Engraftment of hESC-derived CD34+ cells in sheep after in-utero transplantation

Flow cytometric analysis of in vivo multi-lineage differentiation potential of hESC-derived CD34+ cells in comparison to cord blood (CB)-CD34+ cells in peripheral blood after in-utero transplantation. The lymphoid lineage with B cells (CD20), T cells (CD3), NK cells (CD16), and the myeloid lineage with neutrophils (CD15) and monocytes (CD14) were analyzed from animals transplanted with hESC-derived CD34+ cells (# 2786, 2787, and 2788) and CB-CD34+ cells (# 2791 and 2792) at 10 weeks post-transplantation. Animal with no CD34+ transplantation was used as control.

Figure 5. Cross sections of bone from animal transplanted with hESC-derived CD34+ cells

Bone marrow of sheep transplanted with hESC-derived CD34+ cells were stained with anti-human nuclei antibody (green) and anti-CD45 antibody (red). Cell nuclei were stained with DAPI (blue). Microscope images are for the green and blue channels (A), red and blue channels (B), and tri-color (C). While most of the CD45+ cells are endogenous to the sheep, transplanted human cells which are dual positive for CD45 (red) and human nuclei (green) are evident. Dark area represents cross sections of blood vessels in the BM. Photomicrographs were taken on an Olympus Fluoview FV1000 confocal microscope with UPlanFLN 40×1.30 numeric aperture oil objective lens, using FV10-ASW version 01.05.00.14 software (Olympus America Inc., Melville, NY, USA). Images were processed using Adobe Photoshop, version CS5.