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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

Schematic representation of preparation of the transplanted vascular cells differentiated from human ES cells.
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Figure 1: Schematic representation of preparation of the transplanted vascular cells differentiated from human ES cells.

Mentions: Maintenance of human ES cell line (HES-3) was described previously [10]. We plated small human ES colonies on OP9 feeder layer to induce differentiation into ECs and MCs [10]. On day 10 of differentiation, VE-cadherin+VEGF-R2+TRA-1- cells were sorted with a fluorescence activator cell sorter (FACSaria; Becton Dickinson). Monoclonal antibody for VEGF-R2 was labeled with Alexa-647 (Molecular Probes). Monoclonal antibody for TRA1-60 (Chemicon) was labeled with Alexa-488 (Molecular Probes) and anti VE-cadherin (BD Biosciecnces) antibody was labeled with Alexa 546 (Molecular Probes). After sorting the VE-cadherin+VEGFR-2+TRA-1- cells on day 10 of differentiation, we cultured them on type IV collagen-coated dishes (Becton Dickinson) with MEM in the presence of 10% fetal calf serum (FCS) and 50 ng/ml human VEGF165 (Peprotech) and expanded these cells. After five passages in culture (= approximately 30 days after the sorting), we obtained the expanded cells as a mixture of ECs and MCs derived from human ES cells (hES-ECs+MCs). The cell mixture was composed of almost the same number of ECs and MCs. We resorted the VE-cadherin+ cells from these expanded cells to obtain ECs for transplantation (Figure 1). The ECs derived from human ES cells (hES-ECs) were labeled with CM-Dil (Molecular Probes) before the transplantation.


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)

Schematic representation of preparation of the transplanted vascular cells differentiated from human ES cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567291&req=5

Figure 1: Schematic representation of preparation of the transplanted vascular cells differentiated from human ES cells.
Mentions: Maintenance of human ES cell line (HES-3) was described previously [10]. We plated small human ES colonies on OP9 feeder layer to induce differentiation into ECs and MCs [10]. On day 10 of differentiation, VE-cadherin+VEGF-R2+TRA-1- cells were sorted with a fluorescence activator cell sorter (FACSaria; Becton Dickinson). Monoclonal antibody for VEGF-R2 was labeled with Alexa-647 (Molecular Probes). Monoclonal antibody for TRA1-60 (Chemicon) was labeled with Alexa-488 (Molecular Probes) and anti VE-cadherin (BD Biosciecnces) antibody was labeled with Alexa 546 (Molecular Probes). After sorting the VE-cadherin+VEGFR-2+TRA-1- cells on day 10 of differentiation, we cultured them on type IV collagen-coated dishes (Becton Dickinson) with MEM in the presence of 10% fetal calf serum (FCS) and 50 ng/ml human VEGF165 (Peprotech) and expanded these cells. After five passages in culture (= approximately 30 days after the sorting), we obtained the expanded cells as a mixture of ECs and MCs derived from human ES cells (hES-ECs+MCs). The cell mixture was composed of almost the same number of ECs and MCs. We resorted the VE-cadherin+ cells from these expanded cells to obtain ECs for transplantation (Figure 1). The ECs derived from human ES cells (hES-ECs) were labeled with CM-Dil (Molecular Probes) before the transplantation.

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