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Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions.

Uenishi G, Theisen D, Lee JH, Kumar A, Raymond M, Vodyanik M, Swanson S, Stewart R, Thomson J, Slukvin I - Stem Cell Reports (2014)

Bottom Line: The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs).However, the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells.Here, we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC), an extracellular matrix protein associated with HSC niches, strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders, including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI 53792, USA.

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Major Subsets of VEC+ Cells Generated after 5 Days of Differentiation of H1 hESCs in Chemically Defined Conditions on ColIV and TenC(A) Flow-cytometric analysis demonstrates major subsets of VEC+ progenitors generated after 5 days of hESC culture in chemically defined conditions on ColIV and TenC. Lower dot plots show VEC+-gated cells.(B) Percentages of VEC+ cells and subsets generated in ColIV and TenC cultures. Error bars are mean ± SE from at least three experiments (∗p < 0.01).(C) Endothelial and hematopoietic potential of day 5 VEC+ subsets. Progenitor subsets sorted and cultured either in endothelial conditions with subsequent tube formation assay or on OP9 with immunofluorescent and flow-cytometry results after 7 days. Dot plots show expression of VEC and CD43 in TRA-1-85+-gated human cells. Scale bars, 100 μM.(D) CFC potential of an isolated VEC+ subset in serum-free clonogenic medium containing hematopoietic cytokines and FGF2. Error bars are mean ± SE from three experiments (∗p < 0.01). Scale bar represents 100 μm. VEC was visualized using a secondary antibody conjugated to DyLight 488, and CD43 was visualized using a secondary antibody conjugated to DyLight 594.See also Figures S3 and S4.
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fig4: Major Subsets of VEC+ Cells Generated after 5 Days of Differentiation of H1 hESCs in Chemically Defined Conditions on ColIV and TenC(A) Flow-cytometric analysis demonstrates major subsets of VEC+ progenitors generated after 5 days of hESC culture in chemically defined conditions on ColIV and TenC. Lower dot plots show VEC+-gated cells.(B) Percentages of VEC+ cells and subsets generated in ColIV and TenC cultures. Error bars are mean ± SE from at least three experiments (∗p < 0.01).(C) Endothelial and hematopoietic potential of day 5 VEC+ subsets. Progenitor subsets sorted and cultured either in endothelial conditions with subsequent tube formation assay or on OP9 with immunofluorescent and flow-cytometry results after 7 days. Dot plots show expression of VEC and CD43 in TRA-1-85+-gated human cells. Scale bars, 100 μM.(D) CFC potential of an isolated VEC+ subset in serum-free clonogenic medium containing hematopoietic cytokines and FGF2. Error bars are mean ± SE from three experiments (∗p < 0.01). Scale bar represents 100 μm. VEC was visualized using a secondary antibody conjugated to DyLight 488, and CD43 was visualized using a secondary antibody conjugated to DyLight 594.See also Figures S3 and S4.

Mentions: Because the formation of HVMPs in hPSC/OP9 coculture was closely followed by the development of HE and blood progenitors, we supplemented our cultures with SCF, TPO, IL-6, and IL-3 hematopoietic cytokines in addition to VEGF and FGF2, starting from day 4 of differentiation. Although we noticed that the continuous treatment of cultures with FGF2 and VEGF was sufficient for induction of endothelial progenitors and hematopoietic specification, the addition of hematopoietic cytokines was essential to increase the output of these cells in chemically defined cultures. On day 5 of differentiation in these conditions, we observed the three major subsets of the VEC+ population as identified in a previous study (Choi et al., 2012): VEC+CD235a/CD43−CD73+ (non-HEPs), VEC+CD235a/CD43−CD73− (HEPs), and VEC+CD43/ CD235a+ (AHPs) (Figures 4A and 4B). When these subsets were sorted and plated in endothelial conditions, they all formed a monolayer of VEC-expressing cells with the capacity to uptake AcLDL and form vascular tubes in the tube formation assay, consistent with OP9 coculture (Figure 4C). However, hematopoietic CFC potential was mostly restricted to the VEC+CD43/CD235a+ cells (Figure 4D). Importantly, similar to our previous finding with day 5 VEC + subsets generated in coculture with OP9, the hematopoietic CFC potential of VEC+CD43/CD235a+ cells was detected only in serum-free medium in the presence of FGF2 in addition to hematopoietic cytokines (Figures 4D and S3). This indicates that VEC+CD43/CD235a+ cells are essentially similar to the AHPs identified in hPSC/OP9 coculture (Choi et al., 2012). We previously defined HEPs as VEC+CD43−CD73− cells that lack hematopoietic CFC potential but are capable of acquiring it after culture on OP9 (Choi et al., 2012). To determine whether VEC+CD43−CD73− cells generated in completely chemically defined conditions are similar to HEPs produced in OP9 cocultures, we sorted the day 5 VEC+ subpopulations and cultured them on OP9 as previously described (Choi et al., 2012). In these conditions, the HEPs formed both endothelial and hematopoietic cells with a large number of HE clusters, whereas AHPs formed predominantly hematopoietic cells with few endothelial cells and hematoendothelial clusters. VEC+CD43−CD73+ cells formed exclusively endothelial clusters, consistent with the non-HEP phenotype (Figure 4C). Cultures that differentiated on TenC had a larger population of total CD31+ and VEC+ cells, and thus increased populations of HEPs, non-HEPs, and AHPs compared with cultures differentiated on ColIV (Figures 4A, 4B, and S4).


Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions.

Uenishi G, Theisen D, Lee JH, Kumar A, Raymond M, Vodyanik M, Swanson S, Stewart R, Thomson J, Slukvin I - Stem Cell Reports (2014)

Major Subsets of VEC+ Cells Generated after 5 Days of Differentiation of H1 hESCs in Chemically Defined Conditions on ColIV and TenC(A) Flow-cytometric analysis demonstrates major subsets of VEC+ progenitors generated after 5 days of hESC culture in chemically defined conditions on ColIV and TenC. Lower dot plots show VEC+-gated cells.(B) Percentages of VEC+ cells and subsets generated in ColIV and TenC cultures. Error bars are mean ± SE from at least three experiments (∗p < 0.01).(C) Endothelial and hematopoietic potential of day 5 VEC+ subsets. Progenitor subsets sorted and cultured either in endothelial conditions with subsequent tube formation assay or on OP9 with immunofluorescent and flow-cytometry results after 7 days. Dot plots show expression of VEC and CD43 in TRA-1-85+-gated human cells. Scale bars, 100 μM.(D) CFC potential of an isolated VEC+ subset in serum-free clonogenic medium containing hematopoietic cytokines and FGF2. Error bars are mean ± SE from three experiments (∗p < 0.01). Scale bar represents 100 μm. VEC was visualized using a secondary antibody conjugated to DyLight 488, and CD43 was visualized using a secondary antibody conjugated to DyLight 594.See also Figures S3 and S4.
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fig4: Major Subsets of VEC+ Cells Generated after 5 Days of Differentiation of H1 hESCs in Chemically Defined Conditions on ColIV and TenC(A) Flow-cytometric analysis demonstrates major subsets of VEC+ progenitors generated after 5 days of hESC culture in chemically defined conditions on ColIV and TenC. Lower dot plots show VEC+-gated cells.(B) Percentages of VEC+ cells and subsets generated in ColIV and TenC cultures. Error bars are mean ± SE from at least three experiments (∗p < 0.01).(C) Endothelial and hematopoietic potential of day 5 VEC+ subsets. Progenitor subsets sorted and cultured either in endothelial conditions with subsequent tube formation assay or on OP9 with immunofluorescent and flow-cytometry results after 7 days. Dot plots show expression of VEC and CD43 in TRA-1-85+-gated human cells. Scale bars, 100 μM.(D) CFC potential of an isolated VEC+ subset in serum-free clonogenic medium containing hematopoietic cytokines and FGF2. Error bars are mean ± SE from three experiments (∗p < 0.01). Scale bar represents 100 μm. VEC was visualized using a secondary antibody conjugated to DyLight 488, and CD43 was visualized using a secondary antibody conjugated to DyLight 594.See also Figures S3 and S4.
Mentions: Because the formation of HVMPs in hPSC/OP9 coculture was closely followed by the development of HE and blood progenitors, we supplemented our cultures with SCF, TPO, IL-6, and IL-3 hematopoietic cytokines in addition to VEGF and FGF2, starting from day 4 of differentiation. Although we noticed that the continuous treatment of cultures with FGF2 and VEGF was sufficient for induction of endothelial progenitors and hematopoietic specification, the addition of hematopoietic cytokines was essential to increase the output of these cells in chemically defined cultures. On day 5 of differentiation in these conditions, we observed the three major subsets of the VEC+ population as identified in a previous study (Choi et al., 2012): VEC+CD235a/CD43−CD73+ (non-HEPs), VEC+CD235a/CD43−CD73− (HEPs), and VEC+CD43/ CD235a+ (AHPs) (Figures 4A and 4B). When these subsets were sorted and plated in endothelial conditions, they all formed a monolayer of VEC-expressing cells with the capacity to uptake AcLDL and form vascular tubes in the tube formation assay, consistent with OP9 coculture (Figure 4C). However, hematopoietic CFC potential was mostly restricted to the VEC+CD43/CD235a+ cells (Figure 4D). Importantly, similar to our previous finding with day 5 VEC + subsets generated in coculture with OP9, the hematopoietic CFC potential of VEC+CD43/CD235a+ cells was detected only in serum-free medium in the presence of FGF2 in addition to hematopoietic cytokines (Figures 4D and S3). This indicates that VEC+CD43/CD235a+ cells are essentially similar to the AHPs identified in hPSC/OP9 coculture (Choi et al., 2012). We previously defined HEPs as VEC+CD43−CD73− cells that lack hematopoietic CFC potential but are capable of acquiring it after culture on OP9 (Choi et al., 2012). To determine whether VEC+CD43−CD73− cells generated in completely chemically defined conditions are similar to HEPs produced in OP9 cocultures, we sorted the day 5 VEC+ subpopulations and cultured them on OP9 as previously described (Choi et al., 2012). In these conditions, the HEPs formed both endothelial and hematopoietic cells with a large number of HE clusters, whereas AHPs formed predominantly hematopoietic cells with few endothelial cells and hematoendothelial clusters. VEC+CD43−CD73+ cells formed exclusively endothelial clusters, consistent with the non-HEP phenotype (Figure 4C). Cultures that differentiated on TenC had a larger population of total CD31+ and VEC+ cells, and thus increased populations of HEPs, non-HEPs, and AHPs compared with cultures differentiated on ColIV (Figures 4A, 4B, and S4).

Bottom Line: The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs).However, the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells.Here, we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC), an extracellular matrix protein associated with HSC niches, strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders, including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI 53792, USA.

Show MeSH