Evaluating an Alternative Endothelial Cell Source to Vascularize Engineered Tissue Constructs

A major translational challenge in the fields of therapeutic angiogenesis and regenerative medicine is the need to create functional microvasculature. Cell-based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation. Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are a promising, autologous, alternative cell source. The purpose of this study was to assess whether a potentially autologous endothelial cell (EC) source derived from iPSC-ECs can form the same robust, stable microvasculature as previously documented for other sources of ECs. The endothelial lineage of iPSC-ECs was first characterized as through endothelial marker expression and compared to human umbilical vein endothelial cells (HUVECs). Similarities in endothelial markers were demonstrated and genetic expression profile analysis revealed significant genotypic similarities between the iPSC-ECs and HUVECs. A well-established in vitro assay was utilized, in which endothelial cell-coated (iPSC-ECs or HUVECs) beads were co-embedded with fibroblasts in a 3D fibrin matrix, to assess the iPSC-ECs’ ability to form stable microvessels. iPSC-ECs exhibited a five-fold reduction in capillary network formation compared to HUVECs in this assay. Variation of cell sourcing, lot, cell density, and media formulation demonstrated no differences in iPSC-EC sprouting, eliminating these variables as the underlying cause. Despite quantitative differences, iPSC-ECs demonstrated some characteristics of mature vasculature including hollow lumen formation, pericyte recruitment and association, and deposition of basement membrane components. To determine a cause of the in vitro sprouting attenuation, iPSC-ECs’ capillary morphogenetic mechanisms were identified through chemical inhibition of sprouting and analysis of the expression levels of key proteases. Increasing matrix density reduced sprouting, although this effect was attenuated by distributing the normal human lung fibroblasts (NHLFs) within the 3D matrix. Inhibition of both MMP- and plasmin-mediated fibrinolysis was required to completely block sprouting of both HUVECs and iPSC-ECs. Further analysis revealed MMP-9 expression and activity were significantly lower in iPSC-EC/NHLF co-cultures than in HUVEC/NHLF co-cultures, which may account for the observed deficiencies in angiogenic sprouting of the iPSC-ECs. To investigate if the in vitro attenuation was also an in vivo phenomenon, iPSC-ECs were evaluated for their ability to form functional microvasculature in a well-established in vivo model, in which endothelial cells (iPSC-ECs or HUVECs) were co-injected with fibroblasts and a fibrin matrix into the dorsal flank of severe combined immunodeficiency (SCID) mice. Qualitatively, iPSC-ECs were capable of forming perfused vessels that inosculated with mouse vessels and demonstrated similar vessel morphologies to HUVECs. However, quantitatively, iPSC-ECs exhibited a two-fold reduction in vessel density and a three-fold reduction in the number of perfused vessels compared to HUVECs. Further analysis revealed that the presence of the basement membrane component, type IV collagen, and the mural cell marker, alpha-smooth muscle actin, were significantly lower, roughly 25% and 33% respectively, around iPSC-EC/NHLF vasculature relative to that observed in HUVEC/NHLF implants, suggesting reduced vessel maturity. Collectively, these findings demonstrate that a potentially autologous EC with an unlimited source, specifically iPSC-ECs, has the ability to revascularize tissue and argues for a deeper understanding of iPSC-ECs and their differences to enable the promise and potential of iPSC-ECs for clinical translation.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147627/1/bezenahj_1.pd

Impact of Perinatal Escitalopram Exposure on Adolescent Behavior

Escitalopram (EST), a selective serotonin reuptake inhibitor (SSRI), is an antidepressant frequently used during pregnancy, yet there is limited knowledge of its long-term effects on maternal use. Past studies suggest that maternal exposure may adversely affect the fetus, which may be due to the hyperserotonergic environment caused by the nature of SSRIs during early development. Modifications of the serotonergic system in offspring during certain critical periods of development may result in long-term behavioral and cognitive changes in adulthood. Further, recent human studies have demonstrated that prenatal exposure to SSRIs in humans may increase susceptibility to autism spectrum disorder (ASD) and developmental delays. This study examines behavioral and social changes in adolescent rats exposed to escitalopram during postnatal days (PD) 1-10. Eight nulliparous female Long-Evans rats (n = 4 per exposure group) were injected with 10 mg/kg or in 1ml/kg of saline for 10 days. Male and female adolescent offspring were subsequently tested on a battery of behavioral tasks consisting of open field conflict, saccharin taste neophobia, and social approach tasks. Significate decreases in female social interaction were found, as well a moderate increase in inhibitory, anxiety-like behavior in both males and females in the EST group. These findings suggest the occurrence of profound behavioral modification during critical developmental phases, particularly in the neonatal stage, following maternal exposure to escitalopram

Assessing the ability of human endothelial cells derived from induced‐pluripotent stem cells to form functional microvasculature in vivo

Forming functional blood vessel networks is a major clinical challenge in the fields of tissue engineering and therapeutic angiogenesis. Cell‐based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation. Induced‐pluripotent stem cell‐derived endothelial cells (iPSC‐ECs) are a promising, potentially autologous, alternative cell source. However, it is unclear whether iPSC‐ECs form the same robust microvasculature in vivo documented for other EC sources. In this study, we utilized a well‐established in vivo model, in which ECs (iPSC‐EC or human umbilical vein endothelial cells [HUVEC]) were coinjected with normal human lung fibroblasts (NHLFs) and a fibrin matrix into the dorsal flank of severe combined immunodeficiency mice to assess their ability to form functional microvasculature. Qualitatively, iPSC‐ECs were capable of vessel formation and perfusion and demonstrated similar vessel morphologies to HUVECs. However, quantitatively, iPSC‐ECs exhibited a two‐fold reduction in vessel density and a three‐fold reduction in the number of perfused vessels compared with HUVECs. Further analysis revealed the presence of collagen‐IV and α‐smooth muscle actin were significantly lower around iPSC‐EC/NHLF vasculature than in HUVEC/NHLF implants, suggesting reduced vessel maturity. Collectively, these results demonstrate the need for increased iPSC‐EC maturation for clinical translation to be realized.Forming functional blood vessel networks is a major clinical challenge in the fields of tissue engineering and therapeutic angiogenesis. Cell‐based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146963/1/bit26860.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146963/2/bit26860_am.pd