We sought to compare the hematopoietic potential of several hESC lines from different sources. In this analysis we included one human ES cell line reportedly skewed toward mesoderm (HuES8), one toward endoderm (HuES14), one not described (HuES15), the two lines most prevalently used by others for hESC-hematopoietic differentiation, H1 and H9, and another independently-derived hESC cell line, HSF-6 
First, we analyzed the proportion of each hESC line that gave rise to putative hemangioblasts (CD34+
) and hematopoietic progenitor cells (CD34+
) under various passage and differentiation parameters (). We compared the affect of enzymatic (trypsin treatment) versus manual passage on hematopoietic development. To assess the initial commitment to the hematopoietic lineage, we allowed hESCs to differentiate into embryoid bodies (EB) or co-cultured hESCs on an OP9 mouse bone marrow stromal cell monolayer in the absence of lineage skewing cytokines. Consistently, and regardless of cell line, manual passage gave rise to a higher proportion of hESCs differentiating to CD34+
cells in EB culture (). Under the same differentiation conditions, enzymatically passaged hESCs also failed to up-regulate CD45, a marker indicative of hematopoietic commitment. In contrast, under the same differentiation conditions, CD45 was detectable on all the hESC lines maintained through manual passage (). It has been shown that enzymatic passage of hESCs can lead to an increased frequency of karyotype abnormalities 
. Therefore, we also performed karyotype analysis periodically to determine gross karyotypic changes under different culture conditions. Trypsin-passaged HuES8, HuES14, and HuES15 hESC cultures consistently displayed gross karyotype abnormalities (). However, no significant karyotype abnormalities were observed in H1 or H9 trypsin-passaged hESC cultures, nor were any chromosomal abnormalities noted in any of the manually passaged cultures. All hESC lines used throughout the manuscript maintained normal karyotypes with the exception of the top panel of , as noted.
Comparative analysis of hemangioblast development from independently-derived hESC lines.
We next analyzed the propensity of manually passaged hESC lines to generate CD34+ and CD34+CD45+ using a complementary differentiation system. Undifferentiated hESCs were harvested and plated on a monolayer of OP9 mouse bone marrow stromal cells capable of promoting hematopoietic development. Again, CD34 and CD45 expression were monitored by flow cytometry. Overall, all hESC lines consistently gave rise to CD34-expressing cells. However, several differences in the differentiation potential were noted using the two different protocols. Cell-surface CD45 was not detected at any time point on hESCs differentiated in the OP9 co-culture system (, bottom panel and data not shown). In addition, although the H1 line consistently had a higher proportion of CD34+ cells in both differentiation conditions, other hESC lines, specifically H9 and HSF6 generated proportionally more CD34+ cells in the OP9 co-culture system as compared with the EB condition. We also observed that the kinetics of cell-surface CD34 expression differed significantly between hESC lines and the differentiation protocols ().
population contains developmental intermediates capable of giving rise to multiple lineages. To compare the potential of CD34+
cells derived from independent hESC lines, CD34+
cells from EB cultures were enriched by fluorescence activated cell sorting (FACS) and placed into culture with differentiation media containing lineage-promoting cytokines and growth factors that provide hematopoietic- or endothelial-skewing conditions. As expected, under hematopoietic skewing conditions, CD34+
cells differentiated into CD45+
cells (), whereas under endothelial skewing conditions, CD34+
cells up-regulated cell-surface expression of VE-cadherin () 
. Of the lines generating CD45+
cells from CD34+
populations (HuES8, HuES14, H1, and H9), the proportion of CD45+
cells is comparable (.) Whether generation of CD45+
cells in these conditions is due to loss of non-hematopoietic committed cells remains to be seen. We also noticed that the number of CD45+
cells relative to the starting population varied among hESC lines and between experiments (). HuES8, in particular, showed extensive variability in the generation of CD45+
cells between experiments (). This is in contrast to the ability of CD34+
cells from different hESCs to give rise to endothelial cells, which was more consistent from line to line (). Interestingly, H1-derived CD34+
cells generated relatively more CD45+
cells while HSF-6-derived CD34+
cells consistently gave rise to relatively more VE-cadherin+
cells as compared to other hESC lines ().
Skewed hematopoietic vs. endothelial potential from EB-derived CD34+CD45− cells.
It has been shown that hESC-derived hematopoietic progenitor cells differ phenotypically from their in vivo
fetal liver or cord blood counterparts that can easily differentiate into all hematopoietic lineages, being more similar to primitive blood cell progenitors 
. This difference might be one reason for their inability to efficiently generate all the blood lineages in vitro
. To examine the possible differences among hematopoietic progenitors, we assessed the cell-surface expression of a cohort of markers indicative of hematopoietic differentiation state and maturity. Based on CD34, CD31, and CD45 expression, hESC-derived cells were more similar to CD34+
human fetal liver cells, whereas the majority of cord blood CD34+
cells expressed CD45+
cells (). Since the fetal liver and cord blood CD34+
cells have similar lymphoid lineage differentiation potential, and the fetal liver CD34+ cells resemble hESC-derived CD34+ cells, these markers alone cannot distinguish the in vitro
differentiation capacity of CD34+
hESC-derived hematopoietic progenitor cells are phenotypically and developmentally distinct from in vivo hematopoietic precursors.
Co-culture of hematopoietic progenitors on the mouse bone marrow stromal cell line, OP9, is known to support lymphocyte differentiation from a number of human hematopoietic progenitor populations 
. Therefore, we followed the differentiation steps of hESC-derived CD34+
cells on an OP9 monolayer by analyzing the expression of a lymphocyte commitment marker, CD7, by flow cytometry. As shown in , lines HuES8, HuES14, HuES15, and H1 gave rise to a small population of CD7+
cells. In contrast, H9 or HSF6-derived CD34+ cells did not produce appreciable CD7+
cells in OP9 co-culture, and overall, lymphoid progenitor yield was low among lines (). In addition, we analyzed the ability of hESC-derived CD34+
cells to differentiate into T cells. To test T lineage differentiation, we co-cultured hESC-derived CD34+
cells on OP9 stromal cells that express human delta-like 1 Notch ligand (OP9-DL1). This system has been shown to support T lineage differentiation from a variety of mouse and human progenitor cell sources 
. Both human fetal liver and cord blood CD34+
cells generate a significant populations of cells co-expressing CD7 and CD1a marking T lineage commitment within 14 days of co-culture initiation (). In contrast, no CD7+
T cell progenitors were seen in cultures with hESC-derived CD34+
cells (), despite the ability of the same co-culture system to support further differentiation of CD34+
fetal liver progenitors to CD4+
-expressing T lineage cells ().
Several groups have attempted similar differentiation of T lineage cells from hESC-derived progenitor cells with limited success in vitro
. The only exception was a recent report that purportedly found a CD34+
population in a structurally distinct “hematopoietic zone”, which can be differentiated into CD4+
T cells by co-culturing with OP9-DL1 cells 
. Despite extensive search under microscopes, however, we could not detect any “hematopoietic zones” as described in our hESC/OP9-DL1 co-cultures. We also analyzed expression of CD43 in CD34+
cells differentiating in the presence of OP9-DL1 cells. In contrast to cord blood CD34+
cells that did generate T lineage cells and express CD43, no expression of CD43 was detected by flow cytometry on differentiating hESC-derived CD34+