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Transplantation of vascular cells derived from human embryonic stem cells contributes to vascular regeneration after stroke in mice.

Oyamada N, Itoh H, Sone M, Yamahara K, Miyashita K, Park K, Taura D, Inuzuka M, Sonoyama T, Tsujimoto H, Fukunaga Y, Tamura N, Nakao K - J Transl Med (2008)

Bottom Line: We examined the potential of vascular cells derived from human ES cells to contribute to vascular regeneration and to provide therapeutic benefit for the ischemic brain.Transplanted ECs were successfully incorporated into host capillaries and MCs were distributed in the areas surrounding endothelial tubes.Transplantation of ECs and MCs derived from undifferentiated human ES cells have a potential to contribute to therapeutic vascular regeneration and consequently reduction of infarct area after stroke.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan. kanu@kuhp.kyoto-u.ac.jp

ABSTRACT

Background: We previously demonstrated that vascular endothelial growth factor receptor type 2 (VEGF-R2)-positive cells induced from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) and these vascular cells construct blood vessel structures in vitro. Recently, we have also established a method for the large-scale expansion of ECs and MCs derived from human ES cells. We examined the potential of vascular cells derived from human ES cells to contribute to vascular regeneration and to provide therapeutic benefit for the ischemic brain.

Methods: Phosphate buffered saline, human peripheral blood mononuclear cells (hMNCs), ECs-, MCs-, or the mixture of ECs and MCs derived from human ES cells were intra-arterially transplanted into mice after transient middle cerebral artery occlusion (MCAo).

Results: Transplanted ECs were successfully incorporated into host capillaries and MCs were distributed in the areas surrounding endothelial tubes. The cerebral blood flow and the vascular density in the ischemic striatum on day 28 after MCAo had significantly improved in ECs-, MCs- and ECs+MCs-transplanted mice compared to that of mice injected with saline or transplanted with hMNCs. Moreover, compared to saline-injected or hMNC-transplanted mice, significant reduction of the infarct volume and of apoptosis as well as acceleration of neurological recovery were observed on day 28 after MCAo in the cell mixture-transplanted mice.

Conclusion: Transplantation of ECs and MCs derived from undifferentiated human ES cells have a potential to contribute to therapeutic vascular regeneration and consequently reduction of infarct area after stroke.

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Related in: MedlinePlus

Characterization of the transplanted vascular cells derived from human ES cells (HES-3). A, Flow cytometric analysis of VE-cadherin and VEGF-R2 expression on human ES cells during differentiation on an OP9 feeder layer. VE-cadherin+VEGF-R2+TRA-1- cells are indicated by the boxed areas. B, Morphology of the VE-cadherin+ cells (= hES-ECs) resorted from expanded VE-cadherin+VEGF-R2+TRA-1- cells at 5th passage. C, Immunostaining for human PECAM-1 (brown) of hES-ECs. D, Morphology of the expanded VE-cadherin+VEGF-R2+TRA-1- cells at 5th passage (= hES-ECs+MCs). E, Double immunostaining for human PECAM-1 (brown) and αSMA (purple) on hES-ECs+MCs. F, Morphology of the cells (= hES-MCs) expanded from VE-cadherin-VEGF-R2+TRA-1- cells on day 10 of differentiation with PDGF-BB and 1% FCS up to 5th passage. G, Immunostaining for αSMA (brown) of hES-MCs. H-I, Immunostaining for αSMA (green) and calponin (red) of hAoSMCs (H) and hES-MCs (I). Scale bar: 50 μm.
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Figure 2: Characterization of the transplanted vascular cells derived from human ES cells (HES-3). A, Flow cytometric analysis of VE-cadherin and VEGF-R2 expression on human ES cells during differentiation on an OP9 feeder layer. VE-cadherin+VEGF-R2+TRA-1- cells are indicated by the boxed areas. B, Morphology of the VE-cadherin+ cells (= hES-ECs) resorted from expanded VE-cadherin+VEGF-R2+TRA-1- cells at 5th passage. C, Immunostaining for human PECAM-1 (brown) of hES-ECs. D, Morphology of the expanded VE-cadherin+VEGF-R2+TRA-1- cells at 5th passage (= hES-ECs+MCs). E, Double immunostaining for human PECAM-1 (brown) and αSMA (purple) on hES-ECs+MCs. F, Morphology of the cells (= hES-MCs) expanded from VE-cadherin-VEGF-R2+TRA-1- cells on day 10 of differentiation with PDGF-BB and 1% FCS up to 5th passage. G, Immunostaining for αSMA (brown) of hES-MCs. H-I, Immunostaining for αSMA (green) and calponin (red) of hAoSMCs (H) and hES-MCs (I). Scale bar: 50 μm.

Mentions: We induced differentiation of human ES cells in an in vitro two-dimensional culture on OP9 stromal cell line and examined the expression of VEGF-R2, VE-cadherin and TRA-1 during the differentiation. While the population of VE-cadherin+VEGF-R2+TRA-1- cells was not detected (< 0.5%) before day 8 of differentiation, it emerged and accounted for about 1–2% on day10 of differentiation (Figure 2A). As we previously reported, these VE-cadherin+VEGF-R2+TRA-1- cells on day 10 of differentiation were also positive for CD34, CD31 and eNOS [10]. Therefore, we used the term 'eEC' for these ECs in the early differentiation stage. We sorted and expanded these eECs in vitro. These eECs were cultured in the presence of VEGF and 10% FCS and expanded by about 85-fold after 5 passages. The expanded cells at 5th passage were constituted with two cell fractions. One of these cells was VE-cadherin+ cells (35–50%), which were positive for other endothelial markers, including, CD31 (Figure 2B–E) and CD34 [10], indicating that cell differentiation stage had been retained. The other was VE-cadherin- cells (50–65%), which were positive for αSMA and considered to differentiate into MCs (Figure 2D–E). We sorted the fraction of VE-cadherin-VEGF-R2+TRA-1- cells, which appeared on day 8 of differentiation and were positive for platelet derived growth factor receptor type β (PDGFR-β) [10], and expanded these cells for induction to MC in the presence of PDGF-BB and 1% FCS. At passage 5, all of the expanded cells effectively differentiated into αSMA-positive MCs (Figure 2F–G).


Transplantation of vascular cells derived from human embryonic stem cells contributes to vascular regeneration after stroke in mice.

Oyamada N, Itoh H, Sone M, Yamahara K, Miyashita K, Park K, Taura D, Inuzuka M, Sonoyama T, Tsujimoto H, Fukunaga Y, Tamura N, Nakao K - J Transl Med (2008)

Characterization of the transplanted vascular cells derived from human ES cells (HES-3). A, Flow cytometric analysis of VE-cadherin and VEGF-R2 expression on human ES cells during differentiation on an OP9 feeder layer. VE-cadherin+VEGF-R2+TRA-1- cells are indicated by the boxed areas. B, Morphology of the VE-cadherin+ cells (= hES-ECs) resorted from expanded VE-cadherin+VEGF-R2+TRA-1- cells at 5th passage. C, Immunostaining for human PECAM-1 (brown) of hES-ECs. D, Morphology of the expanded VE-cadherin+VEGF-R2+TRA-1- cells at 5th passage (= hES-ECs+MCs). E, Double immunostaining for human PECAM-1 (brown) and αSMA (purple) on hES-ECs+MCs. F, Morphology of the cells (= hES-MCs) expanded from VE-cadherin-VEGF-R2+TRA-1- cells on day 10 of differentiation with PDGF-BB and 1% FCS up to 5th passage. G, Immunostaining for αSMA (brown) of hES-MCs. H-I, Immunostaining for αSMA (green) and calponin (red) of hAoSMCs (H) and hES-MCs (I). Scale bar: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Characterization of the transplanted vascular cells derived from human ES cells (HES-3). A, Flow cytometric analysis of VE-cadherin and VEGF-R2 expression on human ES cells during differentiation on an OP9 feeder layer. VE-cadherin+VEGF-R2+TRA-1- cells are indicated by the boxed areas. B, Morphology of the VE-cadherin+ cells (= hES-ECs) resorted from expanded VE-cadherin+VEGF-R2+TRA-1- cells at 5th passage. C, Immunostaining for human PECAM-1 (brown) of hES-ECs. D, Morphology of the expanded VE-cadherin+VEGF-R2+TRA-1- cells at 5th passage (= hES-ECs+MCs). E, Double immunostaining for human PECAM-1 (brown) and αSMA (purple) on hES-ECs+MCs. F, Morphology of the cells (= hES-MCs) expanded from VE-cadherin-VEGF-R2+TRA-1- cells on day 10 of differentiation with PDGF-BB and 1% FCS up to 5th passage. G, Immunostaining for αSMA (brown) of hES-MCs. H-I, Immunostaining for αSMA (green) and calponin (red) of hAoSMCs (H) and hES-MCs (I). Scale bar: 50 μm.
Mentions: We induced differentiation of human ES cells in an in vitro two-dimensional culture on OP9 stromal cell line and examined the expression of VEGF-R2, VE-cadherin and TRA-1 during the differentiation. While the population of VE-cadherin+VEGF-R2+TRA-1- cells was not detected (< 0.5%) before day 8 of differentiation, it emerged and accounted for about 1–2% on day10 of differentiation (Figure 2A). As we previously reported, these VE-cadherin+VEGF-R2+TRA-1- cells on day 10 of differentiation were also positive for CD34, CD31 and eNOS [10]. Therefore, we used the term 'eEC' for these ECs in the early differentiation stage. We sorted and expanded these eECs in vitro. These eECs were cultured in the presence of VEGF and 10% FCS and expanded by about 85-fold after 5 passages. The expanded cells at 5th passage were constituted with two cell fractions. One of these cells was VE-cadherin+ cells (35–50%), which were positive for other endothelial markers, including, CD31 (Figure 2B–E) and CD34 [10], indicating that cell differentiation stage had been retained. The other was VE-cadherin- cells (50–65%), which were positive for αSMA and considered to differentiate into MCs (Figure 2D–E). We sorted the fraction of VE-cadherin-VEGF-R2+TRA-1- cells, which appeared on day 8 of differentiation and were positive for platelet derived growth factor receptor type β (PDGFR-β) [10], and expanded these cells for induction to MC in the presence of PDGF-BB and 1% FCS. At passage 5, all of the expanded cells effectively differentiated into αSMA-positive MCs (Figure 2F–G).

Bottom Line: We examined the potential of vascular cells derived from human ES cells to contribute to vascular regeneration and to provide therapeutic benefit for the ischemic brain.Transplanted ECs were successfully incorporated into host capillaries and MCs were distributed in the areas surrounding endothelial tubes.Transplantation of ECs and MCs derived from undifferentiated human ES cells have a potential to contribute to therapeutic vascular regeneration and consequently reduction of infarct area after stroke.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan. kanu@kuhp.kyoto-u.ac.jp

ABSTRACT

Background: We previously demonstrated that vascular endothelial growth factor receptor type 2 (VEGF-R2)-positive cells induced from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) and these vascular cells construct blood vessel structures in vitro. Recently, we have also established a method for the large-scale expansion of ECs and MCs derived from human ES cells. We examined the potential of vascular cells derived from human ES cells to contribute to vascular regeneration and to provide therapeutic benefit for the ischemic brain.

Methods: Phosphate buffered saline, human peripheral blood mononuclear cells (hMNCs), ECs-, MCs-, or the mixture of ECs and MCs derived from human ES cells were intra-arterially transplanted into mice after transient middle cerebral artery occlusion (MCAo).

Results: Transplanted ECs were successfully incorporated into host capillaries and MCs were distributed in the areas surrounding endothelial tubes. The cerebral blood flow and the vascular density in the ischemic striatum on day 28 after MCAo had significantly improved in ECs-, MCs- and ECs+MCs-transplanted mice compared to that of mice injected with saline or transplanted with hMNCs. Moreover, compared to saline-injected or hMNC-transplanted mice, significant reduction of the infarct volume and of apoptosis as well as acceleration of neurological recovery were observed on day 28 after MCAo in the cell mixture-transplanted mice.

Conclusion: Transplantation of ECs and MCs derived from undifferentiated human ES cells have a potential to contribute to therapeutic vascular regeneration and consequently reduction of infarct area after stroke.

Show MeSH
Related in: MedlinePlus