To asses the T-cell potential of EB-derived cells we used the H1 hESC line transduced with a EGFP-expressing lentiviral vector [9
]. H1 cells were differentiated in EBs according to the protocol of Chadwick et al. [3
], with the exception of an altered cytokine differentiation cocktail composed of BMP-4, stem cell factor (SCF), and Flt-3L (). The EBs generated in this way exhibited a typical appearance and maintained the expression of EGFP throughout the culture period (). Importantly, this protocol resulted in the development of CD34+ cells, most of which also expressed markers associated with early hematopoietic progenitors, such as CD45, CD43, and CD133 (). CD38, a marker of differentiating hematopoietic progenitors, was also coexpressed on a subset of CD34+ cells. Some of the CD34+ cells also expressed CD184 (CXCR4), a known chemokine receptor critical for progenitor cell migration during thymopoiesis [16
] (). To better define the potential of the EB system to support hematopoietic differentiation, we performed kinetic studies of hematopoietic marker expression and hematopoietic progenitor formation (). The expression of CD34 was detectable by flow cytometry at the earliest time point tested (day 8). However, expression of CD45 lagged and was not observed until day 15 (), indicating that the hematopoietic activity detected in EB cultures at earlier times (described below) precedes CD45 expression. We then assayed the hematopoietic potential of cells cultured as EBs for up to 39 days in a standard methylcellulose-based colony-forming assay. As shown in , the first hematopoietic activity was associated with day 10 of culture. Hematopoietic activity continued to increase until two and half weeks of EB culture, although some myeloid progenitors persisted up to day 39. At earlier time points erythroid colony-forming potential of EB-derived cells was observed, but this diminished over time and was completely lost by day 20. These data suggest that the cytokine combination used in our system supports development of multilineage hematopoietic progenitors.
Figure 1 A diagram of our experimental approach to test the hematopoietic potential of the hESC- and EB-derived cells. EGFP+ hESC were coerced to form EBs and cultured in the presence of cytokines for various periods of time. At different time points the EB-derived (more ...)
Figure 2 Human embryonic stem cell (hESC) differentiation toward hematopoietic lineage in EB cultures. (A): EBs formed by detachment of H1-green fluorescent protein hESC colonies maintained the expression of EGFP throughout the culture period. (B): Cells derived (more ...)
To assess the T lymphoid potential of the EB-derived hematopoietic progenitors we used the SCID-hu mouse. In this animal model small pieces of human fetal liver and thymus are implanted under the renal capsule of SCID mice, where they develop into a thymus-like organ, the Thy/Liv implant [13
]. This conjoint organ provides a microenvironment for long-term T-lineage differentiation. Importantly, we and others have previously shown that direct injection of exogenous CD34+ human hematopoietic progenitor cells into Thy/Liv implants in sublethally irradiated SCID-hu mice results in engraftment and T-lymphoid differentiation of the exogenous cells [9
In two independent experiments, we purified CD34+ cells from 8-, 10-, and 12-day EB cultures (experiment 1) or from 12-, 15-, 18-, and 21-day EB cultures (experiment 2) and injected these into Thy/Liv implants of irradiated SCID-hu mice. The implants were biopsied at different time points, starting at 3 weeks postinjection, and analyzed for the presence of EGFP+ thymocytes that would be derived from hESC (; supporting information Table 1
). Engraftment of the hESC-derived EGFP+ cells was seen in 17 of 38 injected animals and ranged from 0.015% to 23.9% of human cells (supporting information Table 1
). Given the inherent variation in implant sizes, it is misleading to use the percentages of EGFP+ cells following progenitor cell transfer as an absolute measure of the reconstitution efficiency in the transferred population. Therefore, we used the frequency of engraftment as a means of assessing T progenitor potential. Our data indicate that the T-cell reconstitution potential increased with the duration of the EB culture up to day 15, at which point we observed a maximum engraftment frequency of 66%. Interestingly, the T-cell potential seemed to be completely lost by day 21 of EB culture, indicating the transient nature of T-cell progenitor development in this system. However, the presence of EGFP+ thymocytes clearly demonstrates the ability of the EB culture system to generate T lymphoid progenitors.
Kinetic and phenotypic assessment of T cell progenitor development in EB cultures using the SCID-hu system
The implants receiving EB-derived progenitors, analyzed 4 weeks postinjection, displayed a distinct population of CD45+ EGFP+ cells, which was not detected in the control animals (). Most of these cells exhibited a CD4/CD8 double-positive phenotype, indicating an early T-cell developmental stage. This population of cells also expressed T-cell markers CD3, CD5, CD7, and TCRαβ at levels comparable to those found on control thymocytes (). CD45+ EGFP+ cells were still present in the same implants 8 –9 weeks postinjection (, right panels). However, at this time their CD4/CD8 profile changed dramatically and consisted mainly of more mature single-positive CD4 or CD8 cells. This marked decrease in the percentage of CD4/CD8 double-positive cells as a function of time suggests limited self-renewal of T-cell progenitors in this system. These data either imply that the T-cell reconstitution by EB-derived progenitors is transient due to a limited self-renewal capacity of the cells or reflect the limited ability of the SCID-hu system to provide access to an environment needed to support continued stem cell renewal and differentiation. However, as later time points were not assessed, it also remains possible that additional waves of thymopoiesis may occur. It would be of considerable interest to directly compare, in future parallel studies, the longevity of T-lymphoid reconstitution between hESC-derived hematopoietic progenitors and those isolated from fetal liver, cord blood, or mobilized peripheral blood to determine whether any qualitative or quantitative differences between these progenitors exist.
Figure 3 In vivo T lymphoid differentiation of EB-derived hematopoietic progenitor cells expressing eGFP. Shown are flow cytometry profiles of cells derived from irradiated SCID-hu Thy/Liv mice 4 weeks (denoted as week 4) and 8 weeks (denoted as week 8) after (more ...)
Figure 4 Phenotypic characterization of human embryonic stem cell (hESC)-derived CD45+/EGFP+ cells. EB-derived CD45+/EGFP+ cells (gated cells in the upper right quadrant of the top left panel, and other top panels) were compared with the control CD45+ cells (gated (more ...)
Our previous studies using OP9 coculture of hESC could not distinguish the phenotype of the T progenitor cells [9
]. This was largely due to low levels of CD34+ and CD45+ cells in these cocultures. Therefore, to further characterize the phenotype of the EB-derived T-cell progenitors, we purified cells from day 17 EB cultures on the basis of their expression of the hematopoietic differentiation markers CD34, CD45, and CD133. Four purified subsets of cells (CD34+/CD45+, CD34+/CD45−, CD34−/CD133+, and CD34−/CD133−) were assayed for their T-lymphoid potential. Either 5 × 104
or 2.5 × 105
cells of each subset were introduced into Thy/Liv implants of SCID-hu mice. As shown in , our data clearly indicate that the T-cell potential of the cells derived from the EBs cultured for 17 days resides exclusively within the CD34+/CD45+ subset of cells. Implants of seven of eight mice injected with this population of cells contained EGFP+ cells, and these cells also exhibited normal thymocyte profiles of CD4 and CD8 expression. In contrast to this, only 1 (injected with 2.5 × 105
purified CD34+/CD45− cells) of the other 14 animals was positive for hESC-derived thymocytes (). These results also demonstrate the dose-dependent nature of the reconstitution assay. In humans, bone marrow-derived T-cell progenitors have been shown to express both CD34 and CD45 (reviewed in [22
]). It appears that the hESC-derived hematopoietic progenitor cells generated in this culture system are more similar to bone marrow-derived progenitors than are those derived from hESC cocultured on OP9 [9
]. We conclude that the EB culture system supplemented with BMP-4, SCF, and Flt-3L promotes development of T-cell progenitors from hESC and that by day 17 these progenitors exclusively reside within the CD34+CD45+ population of cells.
To assess whether normal T-cell receptor rearrangement takes place in hESC-derived cells, we examined the frequency of usage of different Vβ
gene fragments during the process of V(D)J recombination, as another measure of normal thymocyte development. hESC-derived thymocytes from several Thy/Liv implants were sorted on the basis of EGFP expression, pooled together, and subjected to spectratyping as previously described [14
]. Our data clearly establish that all 24 Vβ
families tested have been used for generation of TCRs within the population of hESC-derived EGFP+ thymocytes (). Furthermore, TCR β
-chain CDR3 length distribution in each Vβ
family showed normal gaussian-like distribution, suggesting that the expected deletions and additions of nucleotides at the recombination junctions also take place during the process of V(D)J recombination in these cells. These data establish that hESC-derived thymocytes undergo a normal process of random V(D)J recombination in the thymic implants during T-cell development. This situation was comparable to that observed in the EGFP-control thymocytes (data not shown) and was similar to that reported in peripheral lymphocytes of healthy individuals [23
Finally, to test the ability of EB-derived thymocytes to respond to TCR-mediated signals, we stimulated these cells with antibodies against CD3 and CD28 in vitro. We have shown previously that these costimulating conditions induce the expression of the high-affinity interleukin-2 receptor (CD25) on thymocytes derived from both normal human thymus and from thymocytes obtained from progenitors following OP9 stromal cells cocultures and injections into Thy/Liv implants [9
]. The EB-derived EGFP+ cells from five of five Thy/Liv implants tested responded to the costimulation by expressing CD25 on their surface (), suggesting that the hESC-derived cells were functional and able to respond to TCR-mediated signals.