Hematopoietic Signaling Mechanism Revealed from a Stem/Progenitor Cell Cistrome.

Thousands of cis-elements in genomes are predicted to have vital functions. Although conservation, activity in surrogate assays, polymorphisms, and disease mutations provide functional clues, deletion from endogenous loci constitutes the gold-standard test. A GATA-2-binding, Gata2 intronic cis-element (+9.5) required for hematopoietic stem cell genesis in mice is mutated in a human immunodeficiency syndrome. Because +9.5 is the only cis-element known to mediate stem cell genesis, we devised a strategy to identify functionally comparable enhancers ("+9.5-like") genome-wide. Gene editing revealed +9.5-like activity to mediate GATA-2 occupancy, chromatin opening, and transcriptional activation. A +9.5-like element resided in Samd14, which encodes a protein of unknown function. Samd14 increased hematopoietic progenitor levels/activity and promoted signaling by a pathway vital for hematopoietic stem/progenitor cell regulation (stem cell factor/c-Kit), and c-Kit rescued Samd14 loss-of-function phenotypes. Thus, the hematopoietic stem/progenitor cell cistrome revealed a mediator of a signaling pathway that has broad importance for stem/progenitor cell biology.

Sterile α-motif domain requirement for cellular signaling and survival.

Hundreds of sterile α-motif (SAM) domains have predicted structural similarities and are reported to bind proteins, lipids, or RNAs. However, the majority of these domains have not been analyzed functionally. Previously, we demonstrated that a SAM domain-containing protein, SAMD14, promotes SCF/proto-oncogene c-Kit (c-Kit) signaling, erythroid progenitor function, and erythrocyte regeneration. Deletion of a Samd14 enhancer (Samd14-Enh), occupied by GATA2 and SCL/TAL1 transcription factors, reduces SAMD14 expression in bone marrow and spleen and is lethal in a hemolytic anemia mouse model. To rigorously establish whether Samd14-Enh deletion reduces anemia-dependent c-Kit signaling by lowering SAMD14 levels, we developed a genetic rescue assay in murine Samd14-Enh-/- primary erythroid precursor cells. SAMD14 expression at endogenous levels rescued c-Kit signaling. The conserved SAM domain was required for SAMD14 to increase colony-forming activity, c-Kit signaling, and progenitor survival. To elucidate the molecular determinants of SAM domain function in SAMD14, we substituted its SAM domain with distinct SAM domains predicted to be structurally similar. The chimeras were less effective than SAMD14 itself in rescuing signaling, survival, and colony-forming activities. Thus, the SAMD14 SAM domain has attributes that are distinct from other SAM domains and underlie SAMD14 function as a regulator of cellular signaling and erythrocyte regeneration.

Mechanisms of erythrocyte development and regeneration: implications for regenerative medicine and beyond.

Hemoglobin-expressing erythrocytes (red blood cells) act as fundamental metabolic regulators by providing oxygen to cells and tissues throughout the body. Whereas the vital requirement for oxygen to support metabolically active cells and tissues is well established, almost nothing is known regarding how erythrocyte development and function impact regeneration. Furthermore, many questions remain unanswered relating to how insults to hematopoietic stem/progenitor cells and erythrocytes can trigger a massive regenerative process termed 'stress erythropoiesis' to produce billions of erythrocytes. Here, we review the cellular and molecular mechanisms governing erythrocyte development and regeneration, and discuss the potential links between these events and other regenerative processes.

Human leukemia mutations corrupt but do not abrogate GATA-2 function.

By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous GATA2 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA2 disease mutations commonly disrupt amino acid residues that mediate DNA binding or cis-elements within a vital GATA2 intronic enhancer, suggesting a haploinsufficiency mechanism of pathogenesis. Mutations also occur in GATA2 coding regions distinct from the DNA-binding carboxyl-terminal zinc finger (C-finger), including the amino-terminal zinc finger (N-finger), and N-finger function is not established. Whether distinct mutations differentially impact GATA-2 mechanisms is unknown. Here, we demonstrate that N-finger mutations decreased GATA-2 chromatin occupancy and attenuated target gene regulation. We developed a genetic complementation assay to quantify GATA-2 function in myeloid progenitor cells from Gata2 -77 enhancer-mutant mice. GATA-2 complementation increased erythroid and myeloid differentiation. While GATA-2 disease mutants were not competent to induce erythroid differentiation of Lin-Kit+ myeloid progenitors, unexpectedly, they promoted myeloid differentiation and proliferation. As the myelopoiesis-promoting activity of GATA-2 mutants exceeded that of GATA-2, GATA2 disease mutations are not strictly inhibitory. Thus, we propose that the haploinsufficiency paradigm does not fully explain GATA-2-linked pathogenesis, and an amalgamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex phenotypes of GATA-2-dependent pathologies.

Mechanism governing a stem cell-generating cis-regulatory element.

The unremitting demand to replenish differentiated cells in tissues requires efficient mechanisms to generate and regulate stem and progenitor cells. Although master regulatory transcription factors, including GATA binding protein-2 (GATA-2), have crucial roles in these mechanisms, how such factors are controlled in developmentally dynamic systems is poorly understood. Previously, we described five dispersed Gata2 locus sequences, termed the -77, -3.9, -2.8, -1.8, and +9.5 GATA switch sites, which contain evolutionarily conserved GATA motifs occupied by GATA-2 and GATA-1 in hematopoietic precursors and erythroid cells, respectively. Despite common attributes of transcriptional enhancers, targeted deletions of the -2.8, -1.8, and +9.5 sites revealed distinct and unpredictable contributions to Gata2 expression and hematopoiesis. Herein, we describe the targeted deletion of the -3.9 site and mechanistically compare the -3.9 site with other GATA switch sites. The -3.9(-/-) mice were viable and exhibited normal Gata2 expression and steady-state hematopoiesis in the embryo and adult. We established a Gata2 repression/reactivation assay, which revealed unique +9.5 site activity to mediate GATA factor-dependent chromatin structural transitions. Loss-of-function analyses provided evidence for a mechanism in which a mediator of long-range transcriptional control [LIM domain binding 1 (LDB1)] and a chromatin remodeler [Brahma related gene 1 (BRG1)] synergize through the +9.5 site, conferring expression of GATA-2, which is known to promote the genesis and survival of hematopoietic stem cells.

Epigenetic and genetic mechanisms in red cell biology.

PURPOSE OF REVIEW: Erythropoiesis, in which hematopoietic stem cells (HSCs) generate lineage-committed progenitors that mature into erythrocytes, is regulated by numerous chromatin modifying and remodeling proteins. We will focus on how epigenetic and genetic mechanisms mesh to establish the erythroid transcriptome and how studying erythropoiesis can yield genomic principles. RECENT FINDINGS: Trans-acting factor binding to small DNA motifs (cis-elements) underlies regulatory complex assembly at specific chromatin sites, and therefore unique transcriptomes. As cis-elements are often very small, thousands or millions of copies of a given element reside in a genome. Chromatin restricts factor access in a context-dependent manner, and cis-element-binding factors recruit chromatin regulators that mediate functional outputs. Technologies to map chromatin attributes of loci in vivo, to edit genomes and to sequence whole genomes have been transformative in discovering critical cis-elements linked to human disease. SUMMARY: Cis-elements mediate chromatin-targeting specificity, and chromatin regulators dictate cis-element accessibility/function, illustrating an amalgamation of genetic and epigenetic mechanisms. Cis-elements often function ectopically when studied outside of their endogenous loci, and complex strategies to identify nonredundant cis-elements require further development. Facile genome-editing technologies provide a new approach to address this problem. Extending genetic analyses beyond exons and promoters will yield a rich pipeline of cis-element alterations with importance for red cell biology and disease.

PDGFRß expression and function in fibroblasts derived from pluripotent cells is linked to DNA demethylation.

Platelet-derived growth factor receptor-beta (PDGFRß) is required for the development of mesenchymal cell types, and plays a diverse role in the function of fibroblasts in tissue homeostasis and regeneration. In this study, we characterized the expression of PDGFRß in fibroblasts derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and showed that this expression is important for cellular functions such as migration, extracellular matrix production and assembly in 3D self-assembled tissues. To determine potential regulatory regions predictive of expression of PDGFRß following differentiation from ESCs and iPSCs, we analyzed the DNA methylation status of a region of the PDGFRB promoter that contains multiple CpG sites, before and after differentiation. We demonstrated that this promoter region is extensively demethylated following differentiation, and represents a developmentally regulated, differentially methylated region linked to PDGFRß expression. Understanding the epigenetic regulation of genes such as PDGFRB, and identifying sites of active DNA demethylation, is essential for future applications of iPSC-derived fibroblasts for regenerative medicine.

Defining a cohort of anemia-activated cis elements reveals a mechanism promoting erythroid precursor function.

Acute anemia elicits broad transcriptional changes in erythroid progenitors and precursors. We previously discovered a cis-regulatory transcriptional enhancer at the sterile alpha motif domain-14 enhancer locus (S14E), defined by a CANNTG-spacer-AGATAA composite motif and occupied by GATA1 and TAL1 transcription factors, is required for survival in severe anemia. However, S14E is only 1 of dozens of anemia-activated genes containing similar motifs. In a mouse model of acute anemia, we identified populations of expanding erythroid precursors, which increased expression of genes that contain S14E-like cis elements. We reveal that several S14E-like cis elements provide important transcriptional control of newly identified anemia-inducing genes, including the Ssx-2 interacting protein (Ssx2ip). Ssx2ip expression was determined to play an important role in erythroid progenitor/precursor cell activities, cell cycle regulation, and cell proliferation. Over a weeklong course of acute anemia recovery, we observed that erythroid gene activation mediated by S14E-like cis elements occurs during a phase coincident with low hematocrit and high progenitor activities, with distinct transcriptional programs activated at earlier and later time points. Our results define a genome-wide mechanism in which S14E-like enhancers control transcriptional responses during erythroid regeneration. These findings provide a framework to understand anemia-specific transcriptional mechanisms, ineffective erythropoiesis, anemia recovery, and phenotypic variability within human populations.

Physiological and regenerative functions of sterile-α motif protein-14 in hematopoiesis.

Sterile α-motif domain-14 (Samd14) protein expression increases the regenerative capacity of the erythroid system. Samd14 is transcriptionally upregulated and promotes cell signaling via the receptor tyrosine kinase Kit in a critical window of acute erythroid regeneration. We generated a hematopoietic-specific conditional Samd14 knockout mouse model (Samd14-CKO) to study the role of Samd14 in hematopoiesis. The Samd14-CKO mouse was viable and exhibited no steady-state hematopoietic phenotype. Samd14-CKO mice were hypersensitive to 5-fluorouracil, resulting in more severe anemia during recovery and impaired erythroid progenitor colony formation. Ex vivo, Samd14-CKO hematopoietic progenitors were defective in their ability to form mast cells. Samd14-CKO mast cells exhibited altered Kit/stem cell factor (SCF), IL-3/IL-3R signaling, and less granularity than Samd14-FL/FL cells. Our findings indicate that Samd14 promotes both erythroid and mast cell functions. The Samd14-CKO mouse phenotype exhibits striking similarities to the KitW/W-v mice, which carry Kit mutations resulting in reduced tyrosine kinase-dependent signaling, causing mast cell and erythroid abnormalities. The Samd14-CKO mouse model is a new tool for studying hematologic pathologies involving Kit signaling.

Functional requirements for a Samd14-capping protein complex in stress erythropoiesis.

Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals-involving cooperation between stem cell factor (SCF)/Kit signaling and other signaling inputs-are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the sterile alpha motif (SAM) domain 14 (Samd14) gene increases the regenerative capacity of the erythroid system in a mouse genetic model and promotes stress-dependent Kit signaling. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. We identified a protein-protein interaction between Samd14 and the α- and ß-heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP ß subunit increased erythroid maturation in murine ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/Kit signaling in CD71med spleen erythroid precursors. Given the roles of Kit signaling in hematopoiesis and Samd14 in Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia.

Anemia is a condition in which the body has a shortage of healthy red blood cells to carry enough oxygen to support its organs. A range of factors are known to cause anemia, including traumatic blood loss, toxins or nutritional deficiency. An estimated one-third of all women of reproductive age are anemic, which can cause tiredness, weakness and shortness of breath. Severe anemia drives the release of hormones and growth factors, leading to a rapid regeneration of precursor red blood cells to replenish the supply in the blood. To understand how red blood cell regeneration is controlled, Ray et al. studied proteins involved in regenerating blood using mice in which anemia had been induced with chemicals. Previous research had shown that the protein Samd14 is produced at higher quantities in individuals with anemia, and is involved with the recovery of lost red blood cells. However, it is not known how the Samd14 protein plays a role in regenerating blood cells, or whether Samd14 interacts with other proteins required for red blood cell production. To shed light on these questions, mouse cells exposed to anemia conditions were used to see what proteins Samd14 binds to. Purifying Samd14 revealed that it interacts with the actin capping protein. This interaction relies on a specific region of Samd14 that is similar to regions in other proteins that bind capping proteins. Ray et al. found that the interaction between Samd14 and the actin capping protein increased the signals needed for the development and survival of new red blood cells. These results identify a signaling mechanism that, if disrupted, could cause anemia to develop. They lead to a better understanding of how our bodies recover from anemia, and potential avenues to treat this condition.

Dissecting Regulatory Mechanisms Using Mouse Fetal Liver-Derived Erythroid Cells.

Multipotent hematopoietic stem cells differentiate into an ensemble of committed progenitor cells that produce the diverse blood cells essential for life. Physiological mechanisms governing hematopoiesis, and mechanistic aberrations underlying non-malignant and malignant hematologic disorders, are often very similar in mouse and man. Thus, mouse models provide powerful systems for unraveling mechanisms that control hematopoietic stem/progenitor cell (HSPC) function in their resident microenvironments in vivo. Ex vivo systems, involving the culture of HSPCs generated in vivo, allow one to dissociate microenvironment-based and cell intrinsic mechanisms, and therefore have considerable utility. Dissecting mechanisms controlling cellular proliferation and differentiation is facilitated by the use of primary cells, since mutations and chromosome aberrations in immortalized and cancer cell lines corrupt normal mechanisms. Primary erythroid precursor cells can be expanded or differentiated in culture to yield large numbers of progeny at discrete maturation stages. We described a robust method for isolation, culture, and analysis of primary mouse erythroid precursor cells and their progeny.

Increased transport of acetyl-CoA into the endoplasmic reticulum causes a progeria-like phenotype.

The membrane transporter AT-1/SLC33A1 translocates cytosolic acetyl-CoA into the lumen of the endoplasmic reticulum (ER), participating in quality control mechanisms within the secretory pathway. Mutations and duplication events in AT-1/SLC33A1 are highly pleiotropic and have been linked to diseases such as spastic paraplegia, developmental delay, autism spectrum disorder, intellectual disability, propensity to seizures, and dysmorphism. Despite these known associations, the biology of this key transporter is only beginning to be uncovered. Here, we show that systemic overexpression of AT-1 in the mouse leads to a segmental form of progeria with dysmorphism and metabolic alterations. The phenotype includes delayed growth, short lifespan, alopecia, skin lesions, rectal prolapse, osteoporosis, cardiomegaly, muscle atrophy, reduced fertility, and anemia. In terms of homeostasis, the AT-1 overexpressing mouse displays hypocholesterolemia, altered glycemia, and increased indices of systemic inflammation. Mechanistically, the phenotype is caused by a block in Atg9a-Fam134b-LC3ß and Atg9a-Sec62-LC3ß interactions, and defective reticulophagy, the autophagic recycling of the ER. Inhibition of ATase1/ATase2 acetyltransferase enzymes downstream of AT-1 restores reticulophagy and rescues the phenotype of the animals. These data suggest that inappropriately elevated acetyl-CoA flux into the ER directly induces defects in autophagy and recycling of subcellular structures and that this diversion of acetyl-CoA from cytosol to ER is causal in the progeria phenotype. Collectively, these data establish the cytosol-to-ER flux of acetyl-CoA as a novel event that dictates the pace of aging phenotypes and identify intracellular acetyl-CoA-dependent homeostatic mechanisms linked to metabolism and inflammation.

GATA Factor-Regulated Samd14 Enhancer Confers Red Blood Cell Regeneration and Survival in Severe Anemia.

An enhancer with amalgamated E-box and GATA motifs (+9.5) controls expression of the regulator of hematopoiesis GATA-2. While similar GATA-2-occupied elements are common in the genome, occupancy does not predict function, and GATA-2-dependent genetic networks are incompletely defined. A "+9.5-like" element resides in an intron of Samd14 (Samd14-Enh) encoding a sterile alpha motif (SAM) domain protein. Deletion of Samd14-Enh in mice strongly decreased Samd14 expression in bone marrow and spleen. Although steady-state hematopoiesis was normal, Samd14-Enh-/- mice died in response to severe anemia. Samd14-Enh stimulated stem cell factor/c-Kit signaling, which promotes erythrocyte regeneration. Anemia activated Samd14-Enh by inducing enhancer components and enhancer chromatin accessibility. Thus, a GATA-2/anemia-regulated enhancer controls expression of an SAM domain protein that confers survival in anemia. We propose that Samd14-Enh and an ensemble of anemia-responsive enhancers are essential for erythrocyte regeneration in stress erythropoiesis, a vital process in pathologies, including ß-thalassemia, myelodysplastic syndrome, and viral infection.

The claudin gene family: expression in normal and neoplastic tissues.

BACKGROUND: The claudin (CLDN) genes encode a family of proteins important in tight junction formation and function. Recently, it has become apparent that CLDN gene expression is frequently altered in several human cancers. However, the exact patterns of CLDN expression in various cancers is unknown, as only a limited number of CLDN genes have been investigated in a few tumors. METHODS: We identified all the human CLDN genes from Genbank and we used the large public SAGE database to ascertain the gene expression of all 21 CLDN in 266 normal and neoplastic tissues. Using real-time RT-PCR, we also surveyed a subset of 13 CLDN genes in 24 normal and 24 neoplastic tissues. RESULTS: We show that claudins represent a family of highly related proteins, with claudin-16, and -23 being the most different from the others. From in silico analysis and RT-PCR data, we find that most claudin genes appear decreased in cancer, while CLDN3, CLDN4, and CLDN7 are elevated in several malignancies such as those originating from the pancreas, bladder, thyroid, fallopian tubes, ovary, stomach, colon, breast, uterus, and the prostate. Interestingly, CLDN5 is highly expressed in vascular endothelial cells, providing a possible target for antiangiogenic therapy. CLDN18 might represent a biomarker for gastric cancer. CONCLUSION: Our study confirms previously known CLDN gene expression patterns and identifies new ones, which may have applications in the detection, prognosis and therapy of several human cancers. In particular we identify several malignancies that express CLDN3 and CLDN4. These cancers may represent ideal candidates for a novel therapy being developed based on CPE, a toxin that specifically binds claudin-3 and claudin-4.

A Hierarchical Framework for State-Space Matrix Inference and Clustering.

In recent years, a large number of genomic and epigenomic studies have been focusing on the integrative analysis of multiple experimental datasets measured over a large number of observational units. The objectives of such studies include not only inferring a hidden state of activity for each unit over individual experiments, but also detecting highly associated clusters of units based on their inferred states. Although there are a number of methods tailored for specific datasets, there is currently no state-of-the-art modeling framework for this general class of problems. In this paper, we develop the MBASIC (Matrix Based Analysis for State-space Inference and Clustering) framework. MBASIC consists of two parts: state-space mapping and state-space clustering. In state-space mapping, it maps observations onto a finite state-space, representing the activation states of units across conditions. In state-space clustering, MBASIC incorporates a finite mixture model to cluster the units based on their inferred state-space profiles across all conditions. Both the state-space mapping and clustering can be simultaneously estimated through an Expectation-Maximization algorithm. MBASIC flexibly adapts to a large number of parametric distributions for the observed data, as well as the heterogeneity in replicate experiments. It allows for imposing structural assumptions on each cluster, and enables model selection using information criterion. In our data-driven simulation studies, MBASIC showed significant accuracy in recovering both the underlying state-space variables and clustering structures. We applied MBASIC to two genome research problems using large numbers of datasets from the ENCODE project. The first application grouped genes based on transcription factor occupancy profiles of their promoter regions in two different cell types. The second application focused on identifying groups of loci that are similar to a GATA2 binding site that is functional at its endogenous locus by utilizing transcription factor occupancy data and illustrated applicability of MBASIC in a wide variety of problems. In both studies, MBASIC showed higher levels of raw data fidelity than analyzing these data with a two-step approach using ENCODE results on transcription factor occupancy data.

Cis-regulatory mechanisms governing stem and progenitor cell transitions.

Cis-element encyclopedias provide information on phenotypic diversity and disease mechanisms. Although cis-element polymorphisms and mutations are instructive, deciphering function remains challenging. Mutation of an intronic GATA motif (+9.5) in GATA2, encoding a master regulator of hematopoiesis, underlies an immunodeficiency associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas an inversion relocalizes another GATA2 cis-element (-77) to the proto-oncogene EVI1, inducing EVI1 expression and AML, whether this reflects ectopic or physiological activity is unknown. We describe a mouse strain that decouples -77 function from proto-oncogene deregulation. The -77(-/-) mice exhibited a novel phenotypic constellation including late embryonic lethality and anemia. The -77 established a vital sector of the myeloid progenitor transcriptome, conferring multipotentiality. Unlike the +9.5(-/-) embryos, hematopoietic stem cell genesis was unaffected in -77(-/-) embryos. These results illustrate a paradigm in which cis-elements in a locus differentially control stem and progenitor cell transitions, and therefore the individual cis-element alterations cause unique and overlapping disease phenotypes.

Strategies for Oral Mucosal Repair by Engineering 3D Tissues with Pluripotent Stem Cells.

Significance: Human-induced pluripotent stem cells (iPSC) can be differentiated into patient-specific cells with a wide spectrum of cellular phenotypes and offer an alternative source of autologous cells for therapeutic use. Recent studies have shown that iPSC-derived fibroblasts display enhanced cellular functions suggesting that iPSC may eventually become an important source of stem cells for regenerative therapies. Recent Advances: The discovery of approaches to reprogram somatic cells into pluripotent cells opens exciting avenues for their use in personalized, regenerative therapies. The controlled differentiation of functional cell types from iPSC provides a replenishing source of fibroblasts. There is intriguing evidence that iPSC reprogramming and subsequent differentiation to fibroblast lineages may improve cellular functional properties. Augmenting the biological potency of iPSC-derived fibroblasts may enable the development of novel, personalized stem cell therapies to treat oral disease. Critical Issues: Numerous questions need to be addressed before iPSC-derived cells can be used as a practical oral therapy. This will include understanding why iPSC-derived cells are predisposed towards differentiation pathways along lineages related to their cell of origin, screening iPSC-derived cells to ensure their safety and phenotypic stability and developing engineered, three-dimensional tissue models to optimize their function and efficacy for future therapeutic transplantation. Future Directions: Future research will need to address how to develop efficient methods to deliver and integrate iPSC-derived fibroblasts into the oral mucosa. This will require an improved understanding of how to harness their biological potency for regenerative therapies that are specifically targeted to the oral mucosa.

Cellular reprogramming to reset epigenetic signatures.

The controlled differentiation of induced pluripotent stem cells (iPSC) towards clinically-relevant cell types has benefitted from epigenetic profiling of lineage-specific markers to confirm the phenotype of iPSC-derived cells. Mapping epigenetic marks throughout the genome has identified unique changes which occur in the DNA methylation profile of cells as they differentiate to specific cell types. Beyond characterizing the development of cells derived from pluripotent stem cells, the process of reprogramming cells to iPSC resets lineage-specific DNA methylation marks established during differentiation to specific somatic cell types. This property of reprogramming has potential utility in reverting aberrant epigenetic alterations in nuclear organization that are linked to disease progression. Since DNA methylation marks are reset following reprogramming, and contribute to restarting developmental programs, it is possible that DNA methylation marks associated with the disease state may also be erased in these cells. The subsequent differentiation of such cells could result in cell progeny that will function effectively as therapeutically-competent cell types for use in regenerative medicine. This suggests that through reprogramming it may be possible to directly modify the epigenetic memory of diseased cells and help to normalize their cellular phenotype, while also broadening our understanding of disease pathogenesis.

Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRß, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.