Cellular kinetics of perivascular MSC precursors

Mesenchymal stem/stromal cells (MSCs) and MSC-like multipotent stem/progenitor cells have been widely investigated for regenerative medicine and deemed promising in clinical applications. In order to further improve MSC-based stem cell therapeutics, it is important to understand the cellular kinetics and functional roles of MSCs in the dynamic regenerative processes. However, due to the heterogeneous nature of typical MSC cultures, their native identity and anatomical localization in the body have remained unclear, making it difficult to decipher the existence of distinct cell subsets within the MSC entity. Recent studies have shown that several blood-vessel-derived precursor cell populations, purified by flow cytometry from multiple human organs, give rise to bona fide MSCs, suggesting that the vasculature serves as a systemic reservoir of MSC-like stem/progenitor cells. Using individually purified MSC-like precursor cell subsets, we and other researchers have been able to investigate the differential phenotypes and regenerative capacities of these contributing cellular constituents in the MSC pool. In this review, we will discuss the identification and characterization of perivascular MSC precursors, including pericytes and adventitial cells, and focus on their cellular kinetics: cell adhesion, migration, engraftment, homing, and intercellular cross-talk during tissue repair and regeneration

Cellular kinetics of perivascular MSC precursors

Mesenchymal stem/stromal cells (MSCs) and MSC-like multipotent stem/progenitor cells have been widely investigated for regenerative medicine and deemed promising in clinical applications. In order to further improve MSC-based stem cell therapeutics, it is important to understand the cellular kinetics and functional roles of MSCs in the dynamic regenerative processes. However, due to the heterogeneous nature of typical MSC cultures, their native identity and anatomical localization in the body have remained unclear, making it difficult to decipher the existence of distinct cell subsets within the MSC entity. Recent studies have shown that several blood-vessel-derived precursor cell populations, purified by flow cytometry from multiple human organs, give rise to bona fide MSCs, suggesting that the vasculature serves as a systemic reservoir of MSC-like stem/progenitor cells. Using individually purified MSC-like precursor cell subsets, we and other researchers have been able to investigate the differential phenotypes and regenerative capacities of these contributing cellular constituents in the MSC pool. In this review, we will discuss the identification and characterization of perivascular MSC precursors, including pericytes and adventitial cells, and focus on their cellular kinetics: cell adhesion, migration, engraftment, homing, and intercellular cross-talk during tissue repair and regeneration. © 2013 William C. W. Chen et al

Generation of three dimensional retinal tissue with functional photoreceptors from human iPSCs

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSC) hold great potential for the modeling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light-sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form 3-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment-disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modeling and open possibilities for future therapies

Challenges and strategies for generating therapeutic patient-specific hemangioblasts and hematopoietic stem cells from human pluripotent stem cells

Recent characterization of hemangioblasts differentiated from human embryonic stem cells (hESC) has further confirmed evidence from murine, zebrafish and avian experimental systems that hematopoietic and endothelial lineages arise from a common progenitor. Such progenitors may provide a valuable resource for delineating the initial developmental steps of human hemato-endotheliogenesis, which is a process normally difficult to study due to the very limited accessibility of early human embryonic/fetal tissues. Moreover, efficient hemangioblast and hematopoietic stem cell (HSC) generation from patient-specific pluripotent stem cells has enormous potential for regenerative medicine, since it could lead to strategies for treating a multitude of hematologic and vascular disorders. However, significant scientific challenges remain in achieving these goals, and the generation of transplantable hemangioblasts and HSC derived from hESC currently remains elusive. Our previous work has suggested that the failure to derive engraftable HSC from hESC is due to the fact that current methodologies for differentiating hESC produce hematopoietic progenitors developmentally similar to those found in the human yolk sac, and are therefore too immature to provide adult-type hematopoietic reconstitution. Herein, we outline the nature of this challenge and propose targeted strategies for generating engraftable human pluripotent stem cell-derived HSC from primitive hemangioblasts using a developmental approach. We also focus on methods by which reprogrammed somatic cells could be used to derive autologous pluripotent stem cells, which in turn could provide unlimited sources of patient-specific hemangioblasts and HSC

Development of Hematopoietic, Endothelial and Perivascular Cells from Human Embryonic and Fetal Stem Cells

Studies of hemangioblasts (a common progenitor of hematopoietic and endothelial cells) during human development are difficult due to limited access to early human embryos. To overcome this obstacle, the in vitro approach of using human embryonic stem cells (hESC) and the embryoid body (hEB) system has been invaluable to investigate the earliest events of hematopoietic and endothelial cell formation. Herein, firstly, optimal culture conditions of hEB were determined for differentiation of hESC toward hematopoietic and endothelial cell lineages and then different developmental stages of hEB were characterized for angio-hematopoietic cell markers expression. Day-8 to day-10 hEB included the highest numbers of hematopoietic and endothelial progenitor cells, and CD34+ CD45- day-10 hEB cells were sorted to evaluate their hemangioblastic cell potential. Next, we established an in vivo chick embryo system that allowed sorted candidate hemangioblast populations to proliferate, migrate, and differentiate. Different stages of hEB cells, day-10 hEB cells purified for expression of CD34, and human peripheral blood hematopoietic stem/progenitor cells were examined for their engraftment capacity in chick embryos. Meanwhile, we examined the multi-lineage differentiation potentiality of CD146+ CD34- differentiating hESC. Recently, our group characterized pericytes/perivascular cells, that displayed positive expression of CD146 (and a lack of mature endothelial cell markers) in a variety of human organisms. Differentiating hESC include a CD146+ population that concomitantly expresses endothelial progenitor cell markers (CD31, CD34, CD133, and BB9). CD146+ pericyte-like hESC were tested for their hematopoietic, myogenic, and neurogenic potential. Finally, perivascular cells were obtained from human fetal placenta villi in order to evaluate their myogenic differentiation, migration ability, and mesenchymal stem cell phenotype. Placental villi are exceptionally rich in fetal microvessels; these might prove to be a beneficial source for stem cells that reside within blood vessel walls. Both CD146+ pericytes isolated from freshly dissociated placenta and purified blood vessel vasculature of placenta were observed for their myogenic potential. In summary, this project provided approaches to understand the early events of hematopoiesis using hESC and a chick embryo model, and allowed for observation of the mesenchymal lineage potential of perivascular cells derived from hESC and human fetal placenta

Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development

We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac–like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis

Protocol to generate endothelial cells, pericytes, and fibroblasts in one differentiation round from human-induced pluripotent stem cells

Summary: Here, we present a protocol for differentiating human-induced pluripotent stem cells into three distinct mesodermal cell types: vascular endothelial cells (ECs), pericytes, and fibroblasts. We describe steps for using monolayer serum-free differentiation and isolating ECs (CD31+) and mesenchymal pre-pericytes (CD31−) from a single differentiation set. We then differentiate pericytes into fibroblasts using a commercial fibroblast culture medium. The three cell types differentiated in this protocol are useful for vasculogenesis, drug testing, and tissue engineering applications.For complete details on the use and execution of this protocol, please refer to Orlova et al. (2014).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics