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Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.

Objective: Cellular and molecular changes that occur during the genesis of the hematopoietic system and hematopoietic stem cells in the human embryo are mostly inaccessible to study and remain poorly understood. To address this gap we have exploited the human embryonic stem cell (hESC) system to molecularly characterize the global transcriptomes of the two functionally discreet and phenotypically separable populations of multipotent hematopoietic cells that first appear when hESCs are induced to differentiate on OP9 cells., Materials and Methods: We prepared long serial analysis of gene expression libraries from lin-CD34+CD43+CD45- and lin-CD34+CD43+CD45+ subsets of primitive hematopoietic cells derived in vitro from hESCs, sequenced them to a depth of 200,000 tags and compared their content with similar libraries prepared from highly purified populations of very primitive human fetal liver and cord blood hematopoietic cells., Results: Comparison of libraries obtained from hESC-derived lin-CD34+CD43+CD45- and lin-CD34+CD43+CD45+ revealed differences in their expression of genes associated with myeloid development, cellular biosynthetic processes, and cell-cycle regulation. Further comparisons with analogous data for primitive hematopoietic cells isolated from first-trimester human fetal liver and newborn cord blood showed an apparent similarity between the transcriptomes of the most primitive hESC- and in vivo-derived populations, with the main differences involving genes that regulate HSC self-renewal and homing, chromatin remodeling, AP1 transcription complex genes, and noncoding RNAs., Conclusion: These data suggest that primitive hematopoietic cells are generated from hESCs in vitro by processes similar to those operative during human embryogenesis in vivo, although some differences were also detected.