Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation

Abstract

SummaryIn the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5–9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived.

Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation

“…Vasculogenesis can be disrupted by the deficiency of the Flk-1 gene in rodents (Shalaby et al, 1995). Thus, establishing a vasculogenesis-disabled trait in the host animal and restoring the trait by exogenous cells may be a strategy to overcome composite vasculogenesis of the host-and donor-derived cells (Hamanaka et al, 2018). In this study, we, therefore, examined whether deficiency of the FLK1 ortholog or kinase insert domain receptor (KDR) in pigs can cause the vasculogenesis-disabled phenotype as seen in rodents (Sakurai et al, 2005;Shalaby et al, 1995) and whether the trait can be restored by blastocyst complementation.…”

“…Fetuses with restored vasculogenesis generated by complementation of the PDX1/KDR-KO embryos were chimeric with a systemically higher contribution of exogenous cells. KDR expression is pivotal for the development of multiple tissues that are distributed throughout the whole body, including vascular endothelia, smooth muscles, and hematopoietic cells (Hamanaka et al, 2018;Sakurai et al, 2005;Shalaby et al, 1995). Therefore, dense distribution of exogenous cells throughout the body tissue of the chimeric fetuses may be hardly avoidable when the empty niche of KDR deficiency is to be compensated.…”

“…This approach is endorsed by the notion that in vitro generation of human organs with complex function and structure is extremely difficult (Rashid et al, 2014), and organ development from PSCs in the natural physiological environment of a xenogeneic fetus would be better supported. Recent studies have been demonstrated compelling evidence for blastocyst complementation in rodents by generating organs, such as kidney, brain, vessels, and blood (Goto et al, 2019;Hamanaka et al, 2018;Kobayashi et al, 2010;Matsunari et al, 2013;Nagashima and Matsunari, 2016;Rashid et al, 2014;Usui et al, 2012;Wu et al, 2016;Yamaguchi et al, 2017).…”

“…In recent years, gene knockout (KO) by genome editing has significantly advanced its feasibility and practical applicability in mammals (Gupta and Musunuru, 2014;Tan et al, 2012). Thus, knocking out the master regulator gene of a specific organ has become a straightforward method for generating the dysorganogenetic phenotype (Goto et al, 2019;Hamanaka et al, 2018;Kobayashi et al, 2010;Usui et al, 2012;Vilarino et al, 2017). Direct genome editing for zygotes, however, has been reported to result in mosaic mutations (Vilarino et al, 2017), which hinder obtaining conclusive evidence for blastocyst complementation.…”

A Novel Strategy for Xeno-Regenerative Therapy

“…The renal lineage cells were derived from the injected PSCs, whereas nonrenal lineages such as blood vessels and stromal cells in kidneys were chimeric for both blastocyst cells and PSCs. Recently, mouse PSC-derived vascular endothelial cells were regenerated into Flk-1 knockout mice, lacking a key gene for vascular endothelial development [38]. By simultaneously disrupting Flk-1 and genes required for genesis of the target organ, rejection-free organs could be generated from patient-specific iPSCs.…”