Abstract
As established cell lines, human pluripotent stem cells such as embryonic stem (ES) or induced pluripotent stem (iPS) cells can divide indefinitely while retaining their potential to differentiate into many cell types in culture. The induction of mesoderm formation and hematopoietic differentiation was achieved either via embryoid body (EB) formation by culturing ES cell aggregation in suspension or by co-cultures with mouse stromal cell lines. Traditionally serum factors are also added for mesoderm induction and hematopoietic differentiation. Although hematopoietic progenitors are obtained from multiple lines of human ES cells, the low efficiency and high variability have hindered the progress of using human ES cells as a model for studying human hematopoiesis. Normally <15% of cells obtained from a primary culture expressed CD34 (a marker for endothelial cells and hematopoietic progenitors) and even less for CD45 (a pan-leukocyte marker). To generate maximal output of CD34+CD45+ hematopoietic progenitors, we decided to adopt the serum-free and spin-EB formation method (Ng, Blood, 2005) and systematically improved culture conditions. 3,000 human ES cells were added into each well in 96-well plates and formed an aggregate after centrifugation. BMP4 and bFGF were added at day 1, and VEGF and hematopoietic cytokines was added at day 3–9. VEGF was then withdrawn after day 9. Single EB (occasionally 2) grew in each well. By day 8, small blast (or lymphocyte-) like cells were observed on the edge of EBs.
By day 12–14, we observed the outgrowth of blast cells (in hundreds to thousands) surrounding each EB (Panel A). By FACS analysis (Panel B), we observed nearly 50% of the total cells express CD45 at day 12, and many co-express CD34. The lymphocytelike cells can be easily separately from EBs by passing through a 40-micron strainer, and nearly all the isolated cells express CD45 (and 50–75% of them co-express CD34). We obtained 6 million CD45+ cells from 0.9 million human ES cells 14 days after EB formation. We also observed that the conditional HES1-ER transgene expression further increased the frequency of CD34+CD45+ cells as we observed under a different culture condition (Yu. Cell Stem Cells, 2008). The isolated CD45+ cells formed efficiently hematopoietic colonies in the methylcellulose medium with a frequency of ~58+/− 4 colonies per 3,000 cells, with or without the HES1-ER transgene. We are currently testing in vivo activities of isolated CD45+ cell populations (+/− HES1-ER) in the NOD/SCID/γC−/− mice. We are also testing if this improved and defined method would also be applicable to hematopoietic differentiation of human iPS cells we recently derived (Mali, Stem Cells, 2008). The significantly improved method using defined media in the absence of serum factors or feeder cells warrants further investigation whether it is better and more reproducible to elucidate mechanisms that regulate early human hematopoiesis, and to generate a large quantity of CD34+CD45+ human hematopoietic progenitor cells for various applications.
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The people behind the papers – Masaki Kinoshita, Toshihiro Kobayashi, Hiroshi Nagashima, Ramiro Alberio and Austin Smith
