iPSC-derived cells' hematopoietic surface marker expression, clonogenic potential, and globin expression pattern. (A) Hematopoietic surface marker expression by iPSC-derived cells. iPSCs were induced to undergo hematopoietic differentiation by embryoid body (EB) formation as previously described.10 Briefly, undifferentiated iPSC colonies were harvested off the murine embryonic fibroblast feeders and plated onto ultra-low attachment plates (Corning) in the EB medium supplemented with 0.3% methylcellulose (BD Biosciences) for 14 days. At the end of the EB culture, EBs were transferred to Matrigel-coated tissue culture plates (BD Biosciences) and cultured in the hematopoietic growth and expansion medium supplemented with a combination of growth factors. The adherent cells were harvested after 7 days and single-cell suspensions were prepared by enzymatic treatment (0.05% dispase + 0.05% collagenase IA) followed by repeated pipetting. Surface markers were stained using monoclonal antibodies and analyzed using FACSCalibur. Heterogeneity in the response to directed hematopoietic differentiation regimen by individual iPSC lines is shown. Similar results were obtained during spontaneous EB differentiation (data not shown). (B) Clonogenic potential of iPSC-derived cells. Cells prepared as described were cultured either in complete human methycellulose medium (StemCell Technologies) for colony-forming units–granulocyte macrophages (CFU-GM) or in a serum-free semisolid medium10 for burst-forming unit erythroid (BFU-E). The frequency of hematopoietic colonies was enumerated after 14 days of culture. The clonogenic potential differed from line to line. (C) Erythroid differentiation of iPSC. EBs from iPSCs were dissociated by enzymatic treatment followed by forcing through 20G and then 18G needles. Single-cell suspensions were cultured in serum-free media supplemented with erythroid growth factors as previously described.10 Fresh media were added as needed to maintain cell density at ∼ 0.5 × 106/mL through the 14- to 18-day culture period. Robust erythroid development was observed from iPSC-MHF2-C1, iPSC-OI12-C1, or iPSC-OI12-C4. A representative flow cytometric scatter plot of iPSC-OI12-C4–derived cells is shown. Similar to erythroid cells from hESCs,10 iPSC-derived immature and mature erythroid cells do not express CD45. (D) β-Locus globin mRNA expression by iPSC-derived erythroid cells. mRNA was prepared from iPSC-derived erythroid cells and the mRNA levels of all β-like genes were measured by quantitative real-time polymerase chain reaction. High levels of ϵ and γ globin mRNA were detected. (E) Immunostaining of iPSC-derived erythroid cells. Smears prepared from the erythroid cells following serum free liquid culture were stained with monoclonal antibodies against ϵ, γ, or β/δ globin chains, followed by a fluorescein isothiocyanate–conjugated anti-mouse antibody and counterstained with DAPI. Pictures were taken under a Leica DMLB microscope with a 40× PL Fluotar objective using a RT Slider Spot camera. The majority of the erythroid cells expressed ϵ and γ globins, and very few β-producing cells were observed.
