Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical Sciences-Oncology Center and Institute for NanoBioTechnology, and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218.
The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature's inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.
"The phase image of the vascular network and a close-up image of the vascular vessel cross-section were shown here. Adapted with permission from Ref. . Copyright 2013, United States National Academy of Sciences. "
"hPSC-derived endothelial progenitors and endothelial cells may provide building blocks for the establishment of in vitro disease models for screening and development of drugs to treat these diseases. Functionality of hPSC-derived endothelial cells has been shown using in vitro cell culture platforms and in vivo animal models (Adams et al., 2013; Kusuma et al., 2013; Orlova et al., 2014; Samuel et al., 2013; Wang et al., 2007). Similar to other somatic cells derived from hPSCs, differentiated CD31 + endothelial cells exhibited functional heterogeneity (Rufaihah et al., 2013). "
[Show abstract][Hide abstract] ABSTRACT: Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here, we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable, and endothelial progenitor differentiation was ?-catenin dependent. Furthermore, by clonal analysis, we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent, capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings, formed tube-like structures, imported acetylated low-density lipoprotein, and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/?-catenin signaling as a primary regulator for generating vascular cells from hPSCs.
"Overall, this sequential tri-culture system constitutes a human BBB model constructed entirely from highly scalable sources. Pericytes have previously been cultured for twenty weeks with over forty population doublings46, which demonstrates their proliferative capacity, although ideally in the future, these may also be derived from stem cells47. Similarly, NPCs have long been recognized for their extensive self-renewal capabilities48, leading to a virtually unlimited supply of neurons and astrocytes upon differentiation. "
[Show abstract][Hide abstract] ABSTRACT: Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction, and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm(2)). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.
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