Efficient and Simultaneous Generation of Hematopoietic and Vascular Progenitors from Human Induced Pluripotent Stem Cells

Cell Culture and Differentiation Protocols

Vascular and Hematopoietic Differentiation

hEB generation

Vascular differentiation conditions were adapted from our previously described hEB protocol (9, 10). hESC and fibroblast-hiPSC lines with >95% undifferentiated morphology were expanded in six-well plates until 80–90% confluency. Culture medium was switched to adaptation medium (AM, see Supporting Information Table S2 for detailed medium composition) for 1 day prior to hEB generation (Table 1). On the next day, pluripotent colonies were treated with 1 mL of 2 mg/mL dispase (Sigma, Cat No. 17105-041) in DMEM/F12 for 5 min at 37°C. The dispase solution was removed, and cell clumps were collected by cell scraping (cell scraper, Sarstedt, Newton, NC) with 1 mL of DMEM/F12. Cells were washed twice in DMEM/F12, and disaggregated by gentle pipetting. Cell suspensions were centrifuged (200 g, 5 min, room temperature), and cell pellets were resuspended in AM (at a concentration of 1 to 2 × 10⁶ cells/mL). ESC/hiPSC suspensions of 0.5 mL were gently deposited over 2 mL of semi-solid methylcellulose medium (MM, see Supporting Information Table S2) in ultra-low attachment six-well plates (Corning, Lowell, MA, Cat No. 3471) using a 5 mL syringe (BD, Franklin Lakes, NJ, Cat No. 309603) and a 16 gauge needle (BD, Cat No. 305198) followed by gentle homogenization. Plates were tightly wrapped using Saran Wrap (SC Johnson, Racine, WI) to create semi-hypoxic conditions, and incubated at 37°C. Two days later, each three wells of small aggregated hEB were collected in 40 mL of phosphate buffer saline (PBS, Life Technologies), centrifuged at 300 g for 5 min at room temperature, washed once more in PBS, and re-suspended in liquid differentiation medium (LDM, see Supporting Information Table S2). The hEB/LDM suspension was returned to the original ultra-low attachment plate and reincubated in hypoxic conditions. hEB were collected every 2 days and resuspended in fresh LDM with supplement of growth factors. A detailed description of this protocol is also available in Ref. 15.

Table 1.

Stepwise procedure for the optimized hemato-vascular differentiation of human pluripotent stem cells

DAYS OF DIFFERENTIATION	NAME OF MEDIUM	PURPOSE	CULTURE DIMENSION
Day 0	hESC mediuma	Undifferentiated state	2D
Day 1	Adaptation medium (AM)a	Pre-differentiation	2D
Day 1 to 3	Methylcellulose medium (MM)a	Forming hEBs, mesodermal lineage commitment	3D
Day 3 to 8	Liquid differentiation medium (LDM)a	Differentiate into mesodermal lineage progenitor cells	3D
Day 8 to 15	EGM-21VEGF (25ng/ml)	Stromal vascular and hematopoietic differentiation	2D
Non-adherent cellsb	Methylcellulose hematopoietic colony assay medium	Enumerate hematopoietic progenitor cells	3D
Post sortb	EGM-2	Purify vascular progenitor fractions	2D
Time frame

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^a

For detailed medium composition, see Supplementary Tables 1 and 2

^b

Cells are derived from day 8 EB with additional differentiation in EGM-2 culture

Hematopoietic Methylcellulose CFU Assays

hEB were enzymatically treated to obtain single cell suspensions, as described below (see section “hESC/hiPSC, hEB, and adherent and non-adherent EGM-2 culture-derived hEB cells”), for determination of hematopoietic progenitor frequency (9, 10). Alternatively, emerging floating cells were harvested from VEGF-supplemented EGM-2 cultures at various time points and assayed for hematopoietic potential by methylcellulose CFU assays as previously described (10, 15). Briefly, either 3 × 10⁵ single hEB cells or floating cells were resuspended in 450 µL of StemSpan^® SFEM medium (serum-free expansion medium, StemCell™ Technologies, Vancouver, BC, Canada) and deposited into 4.05 mL of MethoCult^® serum free methylcellulose medium (MethoCult^® SF H4436, StemCell™ Technologies, Cat No. 04436). The 4.5 mL of cell/methylcellulose mixture were gently vortexed and let to sit down for 5 min to avoid air bubbles prior to load into three 35 mm grid dishes (1.5 mL/dish, Nalge Nunc International, Rochester, NY, Cat No. 174926). Hematopoietic colony formation was assayed between 14 to 21 days of culture. Hematopoietic colonies were counted and photographed using an inverted microscope (Eclipse Ti-u, Nikon Instruments Inc.), and harvested for flow cytometry analysis. If blast immature colonies were still predominant after 10 to 14 days of culture in methylcellulose, 0.5 mL of fresh MethoCult culture medium were added to the culture, and the cells were allowed to grow until 3 weeks of differentiation. Hematopoietic CFU efficiency of EGM-2 culture-derived floating cells was compared to hEB cells (Fig. 4B). hEB cells were maintained in LDM medium for matching timepoints of EGM-2 subculture conditions before disaggregation and methylcellulose CFU assay.

Flow Cytometry

Purification of hEB-Derived Angioblast Populations Via Surface Expression of CD31 and CD146

Immunocytostaining of RUNX1

Day 8 hEB were plated onto fibronectin-coated 12-well plates (0.5 to 1 × 10⁵ cells/well, Greiner Bio-one) and in EGM-2 medium supplemented with 25 ng/mL VEGF for 3–5 days in an incubator (5% CO₂, humid atmosphere), as described above. Adherent cells were washed once in PBS and fixed in 1% paraformaldehyde solution (USB Corporation, Cleveland, OH, Cat No. 19943) in PBS for 20–30 min at 4°C. Staining procedure was adapted from (22). Cells were washed twice 5 min at room temperature in Dako wash buffer (Dako, Carpinteria, CA, Cat No. 53006) and incubated for 30 min at room temperature in PBS, 5% goat serum (Sigma Aldrich, Cat No. G9023-10ML), 0.05% Tween20 (Sigma Aldrich, Cat No. 274348) to block unspecific secondary antibody binding. Blocking solution was disposed of and monoclonal mouse anti-human Runx1 antibody (1:50 in blocking solution, Santa Cruz Biotechnology, Santa Cruz, CA, Cat No. SC-101146, Clone DW71) was readily added to the cells and incubated overnight at 4°C. Incubation with Universal Negative Control for Mouse Primary Antibodies (Dako) was used for negative controls. The next day, cells were washed twice for 5 min in Dako buffer and incubated with biotinylated goat anti-mouse antibody (1:500 in blocking solution, Dako, Cat No. E0433) for 1 h at room temperature. After two consecutive washes, cells were incubated with streptavidin-Cy3 (1:500 in PBS, Sigma Aldrich, Cat No. S6402) for 30 min at room temperature. Cells were washed in PBS and nuclear staining was achieved by incubating with DAPI (4′,6-diamidino-2-phenylindole, 1:1,000 in PBS, Life Technologies) for 5 min. All wells were washed prior to observe and photographed using an epifluorescence microscope (Eclipse Ti-u) and NIS-elements BR3.1 software (Nikon Instruments).

Statistical Evaluation

Data are summarized as average ± standard deviation. Statistical evaluation was performed using two-tailed Student’s t-test. P values are indicated in the figure or in the figure legends.

Optimal Hemato-Vascular Differentiation of hiPSC Required Rigorous Prevention of Spontaneous Differentiation

Although fibroblast-derived hiPSC lines can be routinely cultured in standard hESC medium supplemented with 4 ng/mL FGF2 (2), these lines often displayed a higher frequency of spontaneous differentiation in comparison to standard hESC lines grown in our laboratory. Thus, all hemato-endothelial differentiation experiments were initiated by first assuring we started with only the highest quality of undifferentiated pluripotent stem cell culture. hiPSC colonies with poor differentiated morphology were manually depleted from cultures with a micropipette under a phase contrast microscope in a laminar flow hood, and the remainder was enzymatically passaged by collagenase type IV. Alternatively, undifferentiated colonies were manually dissected, and subcloned onto fresh (after overnight seeding) MEF feeders (Supporting Information Fig. 1A). If necessary, high quality undifferentiated hiPSC subcultures were further stabilized with increased concentrations of FGF2 (e.g., 10–20 ng/mL) than normally required for standard hESC culture (e.g., 4–10 ng/mL). Pluripotency marker expression was regularly checked by flow cytometry and only under these rigorous conditions, fibroblast-hiPSC lines with >90%SSEA4⁺ TRA-81⁺ OCT-4⁺ expression could be routinely maintained prior to hEB differentiation (Supporting Information Fig. S1B).

Hemato-Endothelial Marker Expression on Differentiated hEB was Comparable Between Fibroblast-hiPSC and hESC

We previously demonstrated that mesodermal hemato-endothelial (MHE) and hemangioblastic progenitors sequentially emerge from hESC between 6 and 10 days after hEB differentiation in serum-free medium supplemented with BMP4, VEGF, and FGF2/heparan sulfate (BVF2H) (9, 10) in a manner resembling YS-hematopoiesis of the human embryo. To evaluate the comparative kinetics of hiPSC and hESC hemato-endothelial lineage commitment in our hEB-based system, we tested the differentiation capacities of two fibroblast-derived hiPSC lines, IMR90-1 and IMR90-4, which were previously reported to display diminished hemato-endothelial differentiation potential relative to hESC lines (18, 19). We quantitatively compared the hemo-vascular hEB differentiation efficiency of these hiPSC lines to the hESC line H9 (Fig. 1), which we and others have previously shown exhibits vigorous hemato-endothelial differentiation capacity in both OP9 and hEB differentiation systems (9, 10). Single cell-dissociated hEB cells from all three cell lines were analyzed by flow cytometry analysis at various time points during differentiation for surface marker expression of hemato-endothelial (CD34, CD31) and hemangioblastic (CD143/ACE) antigens (10). Both hESC and hiPSC showed similar kinetics (Fig. 2). A kinetic flow cytometry analysis of hEB cells during differentiation revealed that surface expression of TRA-1-60, TRA-1-81 pluripotency markers rapidly decreased between days 1–5 (Fig. 2). However, surface expression of SSEA4 diminished less rapidly as BVF2H differentiation progressed, with more than 80% of hEB cells still expressing this antigen at day 10 of differentiation. Overall, the kinetics, frequencies, and expressions of these surface markers were relatively similar between hESC and fibroblast-iPSC in this optimized differentiation protocol, although IMR90-4 hiPSC differentiation was more consistently comparable to H9 hESC than IMR90-1. We also noted that surface expression of the mesenchymal-mesodermal marker KDR was abundantly expressed in undifferentiated hESC. Interestingly, surface expression of CD146, a known marker of mesenchymal-pericytic lineage was detected in undifferentiated hESC but not in fibroblast-iPSC (Fig. 2).

CD31 and CD146 Identified a Putative Vascular-Pericytic Population

To further identify mesodermal progenitors generated in this system, we next analyzed the hemato-endothelial population generated in EGM-2 culture. We first subdivided CD31⁺ hEB cells by co-expression of CD146, which has been demonstrated to identify pericyte/perivascular cells (23). All four populations (CD31⁺CD146⁻, CD31⁺CD146⁺, CD31⁻CD146⁻, and CD31⁻CD146⁺ cells) were isolated and analyzed for in vitro endothelial functionality (Fig. 3A). CD31⁺CD146⁺ cells possessed the highest in vitro uptake of Dil-Ac-LDL among the sorted populations, thus demonstrating the most robust endothelial potential (Fig. 3B).

Adherent BVF2H-Differentiated hEB Cells Simultaneously Generated Progenitors with Vascular and Hematopoietic Phenotypes in Endothelial EGM-2 Medium

To enhance the expansion and differentiation of hemangioblasts in our hEB-based differentiation system, we modified our BVF2H differentiation protocol by including a subsequent adherent differentiation step in endothelial culture conditions (10). This was accomplished with defined endothelial medium (EGM-2) to further expand large numbers of hEB-derived hemato-endothelial progenitors in an adherent two-dimensional culture system. Since day 8 hEB contained mesodermal and hemangioblasts in our previously described serum-free BVF2H differentiation system (10), disaggregated hEB cells from this differentiation time point were cultured onto fibronectin-coated plates in EGM-2. This culture system robustly produced adherent, heterogeneous populations of hEB-derived cells with endothelial, hematopoietic, stromal, perivascular, and undifferentiated morphologies. Moreover, we routinely observed the emergence of adherent hematopoietic clusters with round, cobblestone morphology arising from cells with endothelial morphology (15, 24) (Fig. 3C, Supporting Information Fig. S3). This was followed by the emergence of viable floating cells expressing the hematopoietic progenitor markers CD34, CD45, and CD143/ACE (Fig. 4A). These hematopoietic colonies expressed the RUNX1, transcription factor, which is critical for generating definitive hematopoietic cells from hemogenic endothelium (25, 26) (Supporting Information Fig. S4). These floating cells were plated in hematopoietic methylcellulose medium, and were demonstrated to possess higher hematopoietic progenitor frequency than hEB cells at the same differentiation stage (Fig. 4B).

Adherent EGM-2 Culture of BVF2H-differentiated hEB Cells Generated Augmented Frequencies of Multipotent CD34⁺CD45⁺ Hematopoietic Progenitors

To evaluate the efficiency of hematopoietic progenitors that emerged in EGM-2 culture conditions, floating cells were analyzed by flow cytometry analysis at sequential time points for hemato-endothelial markers. These analyses showed that CD34⁺CD45⁺ hematopoietic progenitors emerged from H9 and hiPSC in EGM-2 cultures ~1 week following a wave of CD143⁺CD34⁺ putative hemangioblasts (pre-hematopoietic progenitors). To evaluate the hematopoietic progenitor potential of these floating cells, hEB cells from day 10 hEB, or floating cells of 3, 4, or 5 additional days in EGM-2 culture that started from day 8 EB were plated in methylcellulose medium and enumerated for CFU activity. These experiments revealed that in comparison to day 10 hEB cells in native hEB, floating hematopoietic cells from EGM-2 cultures contained at least three-fold greater frequencies of hematopoietic colonies, including multipotent mixed erythro-myeloid CFU (Fig. 4B).

Interestingly, in contrast to previous reports (18, 20), under these conditions IMR90-1 and IMR90-4 hiPSC both generated erythroid and myeloid CFU colonies that were comparable or slightly greater than H9 hESC (Fig. 4B). Fibroblast-iPSC generated higher numbers of erythroid cells or CFU-e compared to H9 produce higher numbers of BFU-e and mixed colonies. Notably, the phenotype of hematopoietic colonies suggested embryonic and not adult type definitive hematopoiesis, as erythroid colonies expressed embryonic (Hb-ε) and fetal (Hb-F) but not adult beta hemoglobins (HbA) (Fig. 4C).

Supplementation of EGM-2 with Additional Hematopoietic Growth Factors Directed the Further Lineage Commitment of Emerging Hematopoietic Progenitors

To further enhance hematopoietic progenitor generation in this novel system, cultures were supplemented further with additional hematopoietic growth factors at hEB and EGM-2 differentiation stages. For example, when Thrombopoietin (TPO) and IL6 were both added to either the hEB differentiation stage (from day 3 to 8), or during the EGM-2 culture period thereafter, slightly higher numbers of floating CD34⁺CD45⁺ were observed from IMR90-1 and IMR90-4 cell line (Fig. 5A). Addition of TPO and EPO into the EGM-2 medium generated robust erythropoiesis [i.e., CD45⁻CD71⁺ and a higher percentage of glycophorin A (CD235a⁺ cells] compared to EMG2 with simple VEGF supplementation [CD45⁺CD71⁺ and minor glycophorin A (CD235a⁺ cells]. These results suggested that supplementing EGM-2 culture system with lineage-directing hematopoietic growth factors provided a highly efficient means of producing large quantities of lineage-committed erythroid, myeloid, and megakaryocytic progeny (Fig. 5B).