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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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Mesodermal Development from H1 hESCs in Chemically Defined Conditions on ColIV and TenCCultures differentiated on ColIV versus TenC for 2, 3, and 4 days in chemically defined conditions.(A and B) Flow-cytometry plots (A) and graphs (B) comparing the percentage of A+P+ primitive mesodermal population on days 2 and 3.(C) Expression of mesoderm lineage genes measured by quantitative PCR and normalized to RPL13A, comparing day 3 P+ cells and day 4 Khi cells.(D) Comparison of the MB/HB colony-forming potential of day 2, day 3, and day 4 cultures.(E and F) Flow-cytometry plots (E) and graphs (F) comparing the percentage of KDRhiCD31− (Khi) HVMP, CD31+, and KDRloCD31− (Klo) populations on day 4 of differentiation.(G) Hematopoietic and endothelial potential of Khi and Klo cells isolated from day 4 differentiated cells after coculture on OP9 for 7 days. Upper panels show flow cytometry of TRA-1-85+-gated human cells and lower panels show immunofluorescence staining of cells from OP9 cocultures with Khi and Klo cells.In (B)–(D) and (G), bars are mean ± SE from at least 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 S1 and S2.
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fig3: Mesodermal Development from H1 hESCs in Chemically Defined Conditions on ColIV and TenCCultures differentiated on ColIV versus TenC for 2, 3, and 4 days in chemically defined conditions.(A and B) Flow-cytometry plots (A) and graphs (B) comparing the percentage of A+P+ primitive mesodermal population on days 2 and 3.(C) Expression of mesoderm lineage genes measured by quantitative PCR and normalized to RPL13A, comparing day 3 P+ cells and day 4 Khi cells.(D) Comparison of the MB/HB colony-forming potential of day 2, day 3, and day 4 cultures.(E and F) Flow-cytometry plots (E) and graphs (F) comparing the percentage of KDRhiCD31− (Khi) HVMP, CD31+, and KDRloCD31− (Klo) populations on day 4 of differentiation.(G) Hematopoietic and endothelial potential of Khi and Klo cells isolated from day 4 differentiated cells after coculture on OP9 for 7 days. Upper panels show flow cytometry of TRA-1-85+-gated human cells and lower panels show immunofluorescence staining of cells from OP9 cocultures with Khi and Klo cells.In (B)–(D) and (G), bars are mean ± SE from at least 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 S1 and S2.

Mentions: During embryonic development, BMP4, Wnt, and TGFβ/Nodal/Activin A signaling pathways are critical for initiating primitive streak formation and subsequent mesoderm development (Gadue et al., 2005; Keller, 2005). It has been shown that the activation of these signaling pathways is essential to induce the expression of brachyury (T) and KDR (Flk-1, VEGFR2), and initiate mesodermal commitment of mouse PSCs and hPSCs (Cerdan et al., 2012; Kennedy et al., 2007; Nostro et al., 2008; Pearson et al., 2008; Pick et al., 2007; Salvagiotto et al., 2011). We found that high concentrations of BMP4 (50 ng/ml) combined with low concentrations of Activin A (15 ng/ml) and a supplement of LiCl (2 mM) consistently induced expression of the mesodermal surface markers APLNR, KDR, and PDGFRα after 2 days of culture of singularized hESCs on ColIV or TenC, as described above. However, these conditions poorly supported cell survival and required the addition of FGF2 and a hypoxic condition (5% O2, 5% CO2) to improve cell viability and output of mesodermal cells. Day 2 mesodermal cells that differentiated in these conditions expressed PDGFRα and APLNR surface markers, i.e., they became A+P+ cells and displayed MB colony-forming potential (Figures 3A and 3C), similar to what was observed for A+P+ mesodermal cells obtained from day 2 hPSCs differentiated in OP9 coculture (Vodyanik et al., 2010). After 2 days of differentiation, we found that only FGF2 and VEGF were sufficient for A+P+ mesoderm to acquire HB potential on day 3 of differentiation. Similarly to their counterparts generated in OP9 coculture, day 3 A+P+ cells that were generated in chemically defined conditions expressed T and MIXL1 primitive streak genes at a high level, as well as FOXF1 and GATA2 lateral plate mesoderm genes (Figure 3C). The pattern of development was similar in cells cultured on ColIV and TenC. However, the TenC cultures produced significantly more A+P+ cells and MB and HB colonies (Figures 3A, 3B, and 3D).


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)

Mesodermal Development from H1 hESCs in Chemically Defined Conditions on ColIV and TenCCultures differentiated on ColIV versus TenC for 2, 3, and 4 days in chemically defined conditions.(A and B) Flow-cytometry plots (A) and graphs (B) comparing the percentage of A+P+ primitive mesodermal population on days 2 and 3.(C) Expression of mesoderm lineage genes measured by quantitative PCR and normalized to RPL13A, comparing day 3 P+ cells and day 4 Khi cells.(D) Comparison of the MB/HB colony-forming potential of day 2, day 3, and day 4 cultures.(E and F) Flow-cytometry plots (E) and graphs (F) comparing the percentage of KDRhiCD31− (Khi) HVMP, CD31+, and KDRloCD31− (Klo) populations on day 4 of differentiation.(G) Hematopoietic and endothelial potential of Khi and Klo cells isolated from day 4 differentiated cells after coculture on OP9 for 7 days. Upper panels show flow cytometry of TRA-1-85+-gated human cells and lower panels show immunofluorescence staining of cells from OP9 cocultures with Khi and Klo cells.In (B)–(D) and (G), bars are mean ± SE from at least 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 S1 and S2.
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fig3: Mesodermal Development from H1 hESCs in Chemically Defined Conditions on ColIV and TenCCultures differentiated on ColIV versus TenC for 2, 3, and 4 days in chemically defined conditions.(A and B) Flow-cytometry plots (A) and graphs (B) comparing the percentage of A+P+ primitive mesodermal population on days 2 and 3.(C) Expression of mesoderm lineage genes measured by quantitative PCR and normalized to RPL13A, comparing day 3 P+ cells and day 4 Khi cells.(D) Comparison of the MB/HB colony-forming potential of day 2, day 3, and day 4 cultures.(E and F) Flow-cytometry plots (E) and graphs (F) comparing the percentage of KDRhiCD31− (Khi) HVMP, CD31+, and KDRloCD31− (Klo) populations on day 4 of differentiation.(G) Hematopoietic and endothelial potential of Khi and Klo cells isolated from day 4 differentiated cells after coculture on OP9 for 7 days. Upper panels show flow cytometry of TRA-1-85+-gated human cells and lower panels show immunofluorescence staining of cells from OP9 cocultures with Khi and Klo cells.In (B)–(D) and (G), bars are mean ± SE from at least 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 S1 and S2.
Mentions: During embryonic development, BMP4, Wnt, and TGFβ/Nodal/Activin A signaling pathways are critical for initiating primitive streak formation and subsequent mesoderm development (Gadue et al., 2005; Keller, 2005). It has been shown that the activation of these signaling pathways is essential to induce the expression of brachyury (T) and KDR (Flk-1, VEGFR2), and initiate mesodermal commitment of mouse PSCs and hPSCs (Cerdan et al., 2012; Kennedy et al., 2007; Nostro et al., 2008; Pearson et al., 2008; Pick et al., 2007; Salvagiotto et al., 2011). We found that high concentrations of BMP4 (50 ng/ml) combined with low concentrations of Activin A (15 ng/ml) and a supplement of LiCl (2 mM) consistently induced expression of the mesodermal surface markers APLNR, KDR, and PDGFRα after 2 days of culture of singularized hESCs on ColIV or TenC, as described above. However, these conditions poorly supported cell survival and required the addition of FGF2 and a hypoxic condition (5% O2, 5% CO2) to improve cell viability and output of mesodermal cells. Day 2 mesodermal cells that differentiated in these conditions expressed PDGFRα and APLNR surface markers, i.e., they became A+P+ cells and displayed MB colony-forming potential (Figures 3A and 3C), similar to what was observed for A+P+ mesodermal cells obtained from day 2 hPSCs differentiated in OP9 coculture (Vodyanik et al., 2010). After 2 days of differentiation, we found that only FGF2 and VEGF were sufficient for A+P+ mesoderm to acquire HB potential on day 3 of differentiation. Similarly to their counterparts generated in OP9 coculture, day 3 A+P+ cells that were generated in chemically defined conditions expressed T and MIXL1 primitive streak genes at a high level, as well as FOXF1 and GATA2 lateral plate mesoderm genes (Figure 3C). The pattern of development was similar in cells cultured on ColIV and TenC. However, the TenC cultures produced significantly more A+P+ cells and MB and HB colonies (Figures 3A, 3B, and 3D).

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