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Efficient Generation of Hematopoietic Precursors and Progenitors from Human Pluripotent Stem Cell Lines



From the July Edition of Stem Cells
By Stuart P. Atkinson

The study of embryonic development of the hematopoietic system has allowed us to uncover many of the key molecular mechanisms that act at the various different stages. Taking such information into consideration, Woods et al from the lab of Inder M. Verma at the Salk Institute for Biological Studies, La Jolla, California, USA have optimized an in vitro differentiation protocol for the generation of precursors of the hematopoietic lineage and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs).

The initial part of the study entailed the optimization of culture conditions for the differentiation of pluripotent cells to the haematopoietic lineage. Expanding on the work of Hong et al, embryoid bodies (EBs) were generated from ESC and grown in mesodermal-stimulating medium with BMP4, leading to a high yield of CD45+ haematopoietic cells, with the addition of TGFB1 marginally increasing haematopoietic yield. Flow cytometry was used to assay the levels of various haematopoietic markers over the differentiation period, utilising CD45 as an early haematopoietic marker and CD43 as a progenitor marker, both of which showed maximal expression at the three week time point, with differentiation efficiency and colony forming unit (CFU) assay counts also at a maximum at this time.

The presence of endothelial lineages as possible indicators of hematopoietic potential via the hemangioblast or hemogenic endothelium was next studied by the addition of VEGF to the differentiation medium and studying the non-haematopoietic endothelial lineage markers CD34+/CD31+/CD45-. As VEGF receptor 1 (VEGFR1) is present on endothelial cells, including hemogenic endothelial cells, it was hypothesized that adding VEGF to the differentiation cultures could increase hemogenic-endothelial cell proliferation and hematopoietic cell output. Interestingly, after treatment with low levels of VEGF only, a high correlation between blood cell output and the presence of endothelial like cells was observed. High levels of VEGF yielded very few haematopoietic cells, which the authors suggest is perhaps due to negative feedback mechanisms occurring at earlier time points during development.

Next, the authors assayed multiple human ESC and iPSC lines for their ability to differentiate towards the blood lineage using the optimized blood differentiation protocol, including HUES3, H1 hESCs and iPSCs derived from cord blood, fibroblasts and genetically corrected Fanconi disease keratinocytes (Raya et al). All cells tested following the optimized 3 week protocol were able to produce high yields of CD45+ cells (up to 84%; on average 41% ± 16% from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. The functional assay for progenitors, the CFU assay, revealed CFU-granulocytes, CFU-macrophages, and burst-forming unit erythroid colonies, including more primitive CFU-granulocytes, erythrocytes, monocytes, and megakaryocytes colonies. In addition to having significant numbers of CD45+, CD11b+, and CD33+ cells in CFU–granulocyte, monocyte type colonies, the erythroid colonies showed a high proportion of CD45-/GlycophorinA+ cells, indicative of erythropoiesis. Importantly, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. However, upon transplantation of haematopoietic progenitors into NOD/SCID/il2rg-/- mice, the engraftment of CD45+ cells diminished to zero at 10 weeks post transplant from an initial peak of 2.1%, suggesting that the cells generated may be short term repopulating cells and that de novo HSCs were not generated.

This study demonstrates a simple effective haematopoietic differentiation protocol for the expansion of pre-blood precursors and hematopoietic progenitors, which this study shows functions well when applied to multiple ESC and iPSC lines. Yet this protocol failed to derive long-term repopulating haematopoietic cells from pluripotent cell types, prompting continued investigation into the additional supportive signals which may be necessary to achieve this aim, either during the differentiation process or following donor cell engraftment. The protocols used in this study, however, represent a solid platform from which to progress and discover those factors required for the generation and expansion of definitive HSCs with long-term repopulating ability.



Woods NB, Parker AS, Moraghebi R, Lutz MK, Firth AL, Brennand KJ, Berggren WT, Raya A, Belmonte JC, Gage FH, Verma IM.
Brief report: efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines.
Stem Cells. 2011 Jul;29(7):1158-64. doi: 10.1002/stem.657.

Hong SH, Werbowetski-Ogilvie T, Ramos-Mejia V, Lee JB, Bhatia M.
Multiparameter comparisons of embryoid body differentiation toward human stem cell applications.
Stem Cell Res. 2010 Sep;5(2):120-30. Epub 2010 Apr 28.

Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells.
Raya A, Rodríguez-Pizà I, Guenechea G, Vassena R, Navarro S, Barrero MJ, Consiglio A, Castellà M, Río P, Sleep E, González F, Tiscornia G, Garreta E, Aasen T, Veiga A, Verma IM, Surrallés J, Bueren J, Izpisúa Belmonte JC.
Nature. 2009 Jul 2;460(7251):53-9. Epub 2009 May 31.