Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells.
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SUBMITTER: Hu S
PROVIDER: S-EPMC4937999 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundEndothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.MethodsWe transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.ResultsWe report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.ConclusionECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.
| S-EPMC7510078 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study, we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic), lin(-)CD34(+)CD43(+)CD45(-) (multipotent), and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal, or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks.
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Project description:This study examined the homing capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and their response to chemotactic gradients of stromal derived factor-1? (SDF). We have previously shown that EC derived from murine pluripotent stem cells can home to the ischemic hindlimb of the mouse. In the current study, we were interested to understand if ECs derived from human induced pluripotent stem cells are capable of homing. The homing capacity of iPSC-ECs was assessed after systemic delivery into immunodeficient mice with unilateral hindlimb ischemia. Furthermore, the iPSC-ECs were evaluated for their expression of CXCR4 and their ability to respond to SDF chemotactic gradients in vitro. Upon systemic delivery, the iPSC-ECs transiently localized to the lungs but did not home to the ischemic limb over the course of 14 days. To understand the mechanism of the lack of homing, the expression levels of the homing receptor, CXCR4, was examined at the transcriptional and protein levels. Furthermore, their ability to migrate in response to chemokines was assessed using microfluidic and scratch assays. Unlike ECs derived from syngeneic mouse pluripotent stem cells, human iPSC-ECs do not home to the ischemic mouse hindlimb. This lack of functional homing may represent an impairment of interspecies cellular communication or a difference in the differentiation state of the human iPSC-ECs. These results may have important implications in therapeutic delivery of iPSC-ECs.
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Project description:Cadmium (Cd) is a harmful heavy metal that results in vascular diseases such as atherosclerosis. Prior evidence revealed that Cd induced endothelial cell (EC) death and dysfunction, supporting that ECs are a primary target of Cd-induced toxicity, and can cause severe pathologies of vascular diseases. However, the underlying mechanisms remain unclear. In this study, we investigated the mechanisms of Cd-induced endothelial toxicity in a human model system of H9 human pluripotent stem cell-derived endothelial cells (H9-ECs). We showed that H9-ECs were susceptible to CdCl2 induction, leading to detrimental changes of cell structure and significantly elevated level of apoptosis. We demonstrated that CdCl2-treated H9-ECs gave rise to a clear EC dysfunction phenotype and significantly differential transcriptomic profile. Signaling pathway analysis revealed that P38 or ERK signaling pathway is critical to cadmium-induced EC apoptosis and dysfunction, and inhibition of P38 or ERK effectively rescued CdCl2-induced endothelial toxicity in H9-ECs. Conclusively, hPSC-ECs can be a reliable model to recapitulate the EC pathological features and transcriptomic profile, which may provide a unique platform for understanding the cellular and molecular mechanisms of Cd-induced endothelial toxicity and for identifying therapeutic drugs for Cd-induced vascular diseases.
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Project description:Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are promising for treatment of vascular diseases. However, hiPSC-ECs purified based on CD31 expression are comprised of arterial, venous, and lymphatic subtypes. It is unclear whether hiPSC-ECs are heterogeneous in nature, and whether there may be functional benefits of enriching for specific subtypes. Therefore, we sought to characterize the hiPSC-ECs and enrich for each subtype, and demonstrate whether such enrichment would have functional significance. The hiPSC-ECs were generated from differentiation of hiPSCs using vascular endothelial growth factor (VEGF)-A and bone morphogenetic protein-4. The hiPSC-ECs were purified based on positive expression of CD31. Subsequently, we sought to enrich for each subtype. Arterial hiPSC-ECs were induced using higher concentrations of VEGF-A and 8-bromoadenosine-3':5'-cyclic monophosphate in the media, whereas lower concentrations of VEGF-A favored venous subtype. VEGF-C and angiopoietin-1 promoted the expression of lymphatic phenotype. Upon FACS purification based on CD31+ expression, the hiPSC-EC population was observed to display typical endothelial surface markers and functions. However, the hiPSC-EC population was heterogeneous in that they displayed arterial, venous, and to a lesser degree, lymphatic lineage markers. Upon comparing vascular formation in matrigel plugs in vivo, we observed that arterial enriched hiPSC-ECs formed a more extensive capillary network in this model, by comparison to a heterogeneous population of hiPSC-ECs. This study demonstrates that FACS purification of CD31+ hiPSC-ECs produces a diverse population of ECs. Refining the differentiation methods can enrich for subtype-specific hiPSC-ECs with functional benefits of enhancing neovascularization.
| S-EPMC3560482 | biostudies-literature
Project description:Endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) provide a new opportunity for mechanistic research on vascular regeneration and drug screening. However, functions of hiPSC-ECs still need to be characterized. The objective of this study was to investigate electrophysiological and functional properties of hiPSC-ECs compared with primary human cardiac microvascular endothelial cells (HCMECs), mainly focusing on ion channels and membrane receptor signaling, as well as specific cell functions. HiPSC-ECs were derived from hiPS cells that were generated from human skin fibroblasts of three independent healthy donors. Phenotypic and functional comparison to HCMECs was performed by flow cytometry, immunofluorescence staining, quantitative reverse-transcription polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), tube formation, LDL uptake, exosome release assays and, importantly, patch clamp techniques. HiPSC-ECs were successfully generated from hiPS cells and were identified by endothelial markers. The mRNA levels of KCNN2, KCNN4, KCNMA1, TRPV2, and SLC8A1 in hiPSC-ECs were significantly higher than HCMECs. AT1 receptor mRNA level in hiPSC-ECs was higher than in HCMECs. AT2 receptor mRNA level was the highest among all receptors. Adrenoceptor ADRA2 expression in hiPSC-ECs was lower than in HCMECs, while ADRA1, ADRB1, ADRB2, and G-protein GNA11 and Gai expression were similar in both cell types. The expression level of muscarinic and dopamine receptors CHRM3, DRD2, DRD3, and DRD4 in hiPSC-ECs were significantly lower than in HCMECs. The functional characteristics of endothelial cells, such as tube formation and LDL uptake assay, were not statistically different between hiPSC-ECs and HCMECs. Phenylephrine similarly increased the release of the vasoconstrictor endothelin-1 (ET-1) in hiPSC-ECs and HCMECs. Acetylcholine also similarly increased nitric oxide generation in hiPSC-ECs and HCMECs. The resting potentials (RPs), ISK1-3, ISK4 and IK1 were similar in hiPSC-ECs and HCMECs. IBK was larger and IKATP was smaller in hiPSC-ECs. In addition, we also noted a higher expression level of exosomes marker CD81 in hiPSC-ECs and a higher expression of CD9 and CD63 in HCMECs. However, the numbers of exosomes extracted from both types of cells did not differ significantly. The study demonstrates that hiPSC-ECs are similar to native endothelial cells in ion channel function and membrane receptor-coupled signaling and physiological cell functions, although some differences exist. This information may be helpful for research using hiPSC-ECs.
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Project description:The multifaceted extracellular milieu presents biochemical and biophysical stimuli that influence stem cell differentiation. Two-dimensional (2D) micropatterned substrates allow the presentation of these cues in spatially defined geometries that have been demonstrated to guide stem cell fate decisions. Leveraging stem cells to reconstruct microvasculature, made up of an inner lining of endothelial cells (ECs) supported by pericytes, is critical to tissue-engineering advances; thus, methods to improve endothelial differentiation efficiency are vital to these efforts. In this study, we examine the hypothesis that the diameter of micropatterned islands influences endothelial differentiation from human induced pluripotent stem cells (hiPSCs). Comparing island diameters of 80, 140, 225 and 500 µm, we found that co-cultures of control ECs and pericytes did not yield variable ratios of cell types; however, when hiPSCs were differentiated toward a bicellular population of ECs and pericytes on these varying micropattern feature sizes, we found that smaller islands promoted EC differentiation efficiency, yielding a derived population composed of 70% ECs, which exhibited a greater sprouting propensity. Differentiation on the largest feature size exhibited a smaller EC yield, similar to that on non-patterned substrates. Taken together, these data demonstrate that micropatterned islands of varying diameters can be used to modulate EC differentiation efficiency. Copyright © 2015 John Wiley & Sons, Ltd.
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Project description:The vascular system is essential for the development and function of all organs and tissues in our body. The molecular signature and phenotype of endothelial cells (EC) are greatly affected by blood flow-induced shear stress, which is a vital component of vascular development and homeostasis. Recent advances in differentiation of ECs from human induced pluripotent stem cells (hiPSC) have enabled development of in vitro experimental models of the vasculature containing cells from healthy individuals or from patients harboring genetic variants or diseases of interest. Here we have used hiPSC-derived ECs and bulk- and single-cell RNA sequencing to study the effect of flow on the transcriptomic landscape of hiPSC-ECs and their heterogeneity. We demonstrate that hiPS-ECs are plastic and they adapt to flow by expressing known flow-induced genes. Single-cell RNA sequencing showed that flow induced a more homogenous and homeostatically more stable EC population compared to static cultures, as genes related to cell polarization, barrier formation and glucose and fatty acid transport were induced. The hiPS-ECs increased both arterial and venous markers when exposed to flow. Interestingly, while in general there was a greater increase in the venous markers, one cluster with more arterial-like hiPS-ECs was detected. Single-cell RNA sequencing revealed that not all hiPS-ECs are similar even after sorting, but exposing them to flow increases their homogeneity. Since hiPS-ECs resemble immature ECs and demonstrate high plasticity in response to flow, they provide an excellent model to study vascular development.
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Project description:Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function, given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus, to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons, using a protocol for motor neuron differentiation, we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes, cell survival, cell fate, and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival, altered neuronal differentiation, and, in particular, synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation.
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Project description:Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet's coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.
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