Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation

Embryoid body (EB) differentiation of murine embryonic stem cells (mESCs) recapitulates many aspects of early mouse embryogenesis.¹  Synchronous differentiation is typically achieved by forming mEBs from mESCs seeded at low cell densities.^2-4  Because human embryonic stem cells (hESCs) survive poorly as single cells,⁵  human EB formation has generally been initiated from enzymatically digested colony pieces of different sizes or from high-density suspension cultures.^6-11  Cocultivation of hESCs with stromal layers, such as OP9 and S17, has successfully induced hematopoietic differentiation,^7,8,12  but these systems are compromised by cell-associated and secreted components derived from the feeder layer and by potential interactions between added factors and the stromal layers.⁷  The weaknesses inherent in the existing protocols highlight the need for a robust stromal- and serum-free culture system for the differentiation of hESCs.

Human embryonic stem cells 2, 3, and 4 (hES2, hES3, and hES4)¹³  and the green fluorescence protein (GFP)–expressing hES3 derivative ENVY¹⁴  were cultured essentially as described.⁵  A detailed protocol for hESC differentiation is provided in the supplemental Document S1 (see the Supplemental Materials link at the top of the online article, at the Blood website). In brief, hESCs were trypsinized into a single-cell suspension, washed in phosphate-buffered saline, and resuspended in serum-free medium (SFM).¹⁵  EB formation was induced by seeding the desired number (300-10 000) of hESCs in 100 μL SFM supplemented with growth factors in each well of 96-well, round-bottomed, low-attachment plates (Nunc, Roskilde, Denmark) and centrifuging the plates at 1500 rpm (478g) for 4 minutes at 4°C to aggregate the cells. After 10 to 12 days, the EBs were transferred to 96-well, flat-bottomed tissue culture plates precoated with gelatin in SFM supplemented with growth factors and were allowed to differentiate further.

In our initial attempts to direct hematopoietic differentiation of hESCs, hEBs were formed from pieces of undifferentiated colonies in the same SFM that we had used to induce mesoderm from mESCs.⁴  However, only the subset of hEBs that developed from larger colony fragments (approximately 500-1000 cells) regularly formed blood cells, suggesting that a minimum number of hESCs was required to generate hEBs that differentiated into mesodermal lineages.

Because it was not possible to reproducibly disaggregate hESC colonies into pieces of exactly the desired size, we attempted to improve the efficiency of hematopoietic differentiation by depositing known numbers of hESCs in round-bottomed, low-adherence, 96-well plates and then aggregating the cells into EBs by centrifugation (Figure 1A). After 8 to 12 days, these so-called spin EBs were transferred to tissue culture–treated plates and differentiated further. The wells were scored for the presence of developing myeloid and hemoglobinized erythroid cells by light microscopy. Visual cell lineage assignments were confirmed by correlating the morphologic appearance of cells in the wells with May-Grünwald-Giemsa–stained cytocentrifuge preparations, flow cytometry, immunocytochemistry, and gene expression analysis in selected cases (Figure 1; Figures S1-S3).

The differentiation process was affected by several parameters. If the hESCs were seeded into flat-bottomed wells, they failed to form large aggregates even after centrifugation of the cells (Figure 1B), and both survival and differentiation were unpredictable. Conversely, deposition of hESCs into low-attachment round-bottomed wells followed by brief centrifugation encouraged cellular association into a close pellet (Figure 1C) from which uniform EBs reproducibly formed and differentiated (see examples in Figure 2). Hematopoietic differentiation was also influenced by the number of cells seeded into each well. In the representative experiment shown in Figure 1D, efficient blood formation required in excess of 500 hESCs per well, and optimum erythropoiesis was observed when 1000 cells were seeded per well. In 5 experiments in which 3000 hESCs were deposited per well, EBs developed in 1112 of 1164 (95.5%) wells, with an average of 95.8% ± 1.6% of wells containing viable EBs and 91.8% ± 7.8% of wells containing blood cells per experiment.

Gene expression analysis of hEBs revealed sequential expression of differentiation stage–related genes, similar to the pattern of gene expression observed in cultures of mouse EBs (Figure 1E).^4,16-18  During hESC differentiation, gradual attenuation of the expression of the stem cell gene OCT4 was observed. The primitive streak genes MIXL1 and BRACHYURY were strongly expressed from day 4 to day 8, indicating a wave of mesendodermal induction. Vascular endothelial growth factor (VEGF) receptor 2 (FLK1/KDR) expression increased and remained at high levels throughout the differentiation period, consistent with the ventral patterning of mesoderm in these cultures, followed at day 10 and day 20 by peak expression of the hematopoietic and endothelial gene surface marker CD34 and the hematopoietic transcription factor RUNX1, respectively.^17,19 

Most EBs generated by this protocol generated large numbers of hemoglobinized or nonhemoglobinized blood cells (Figure 1F-G). May-Grünwald-Giemsa–stained cytospins showed neutrophils, macrophages, and mast cells in wells containing myeloid cells (Figure 1H) and maturing erythroid cells in wells containing overtly hemoglobinized cells (Figure 1I). Although most erythroid cells were nucleated, consistent with a primitive, yolk sac lineage, occasional cells undergoing enucleation were observed, suggesting the presence of definitive erythropoiesis (Figure 1I, inset). Flow cytometry of EBs disaggregated at day 11 showed high proportions of CD34⁺ and CD38⁺ cells (Figure 1J), confirming the efficiency of differentiation. Analysis at day 26 showed that the cultures supported the expansion of hematopoietic mesoderm because most cells expressed either CD45 or Tie-2, marking hematopoietic or endothelial lineages, respectively (Figure 1K).

The reproducibility of the spin EB method was exploited to estimate the frequency of hematopoietic precursors arising from a defined number of hESCs. EBs were formed from 3000 hESCs consisting of variable combinations of wild-type hES3 cells and a GFP-expressing derivative, denoted ENVY.¹⁴  Aggregation of GFP⁺ and GFP^– hESCs led to the formation of chimeric EBs into which GFP⁺ cells were integrated in proportion to their input number (Figure 2A). Analysis of these data demonstrated an excellent correlation (R²  > 0.99 for the 3 experiments shown in Figure 2B) between the number of GFP⁺ input cells and the percentage of EBs forming GFP⁺ blood cells. The very high correlation coefficient is a testament to the reproducible differentiation observed over many hundreds of wells. Using Poisson statistics, the frequency of hematopoietic precursors was estimated at approximately 1 in 500 input cells.

In preliminary experiments examining the development of methylcellulose colony-forming cells (CFCs), we observed CFCs from day 6 spin EBs giving rise to primitive erythroid and macrophage colonies at a frequency of 92 per 10⁴ cells and day 10 CFCs at a frequency of 23 per 10⁴ cells. Taken together, the high frequencies of day 11 CD34⁺ cells and hematopoietic precursors observed in our system compared favorably with the results reported using conventional EB or stromal cocultivation methods for hESC differentiation^6-8,10,12  and were superior to the precursor frequencies reported by others for human EBs differentiated in serum-free cultures.⁷  At a molecular level, the spin EBs passed through a transient in vitro gastrulation stage that antedated the expression of mesodermal genes and the emergence of hematopoietic cells, similar to differentiating mESCs.^4,16-18  The ability to faithfully reproduce early human embryonic differentiation in vitro provides a powerful tool for the molecular and cellular dissection of this previously inaccessible phase.

We believe that modulating medium composition, extracellular matrix substrate, and hESC numbers will enable spin EBs to be readily adapted to the generation of many cell types. In addition, the ability to form chimeric EBs with marked cells, such as ENVY, will permit an estimation of clonogenic frequency in cell types for which simple precursor assays are unavailable.