Source Of Patient-specific Cells Research Articles

Generation of iPSC Cell Lines from Patients with Sex Chromosome Aneuploidies.

Somatic cell reprogramming allows the generation of human induced pluripotent stem cells (iPSCs) from patient's cells. The derived iPSCs provide an unlimited source of patient-specific cells that can be virtually differentiated in any cell of the human body. The generation of iPSCs has important implications for all human medicine fields, as they can be used for drug discovery, regenerative medicine, and developmental studies. Klinefelter Syndrome (KS) is the most common chromosome aneuploidy in males. KS is typically characterized by a 47,XXY karyotype, representing 80-90% of KS patients. In rare cases, high-grade sex chromosome aneuploidies (SCAs), 48,XXXY; 48,XXYY; 49,XXXXY, are also observed in males. Since the advent of the reprogramming technique, a few KS-iPSCs have been described. Here, we detail the methodology for generating primary fibroblasts from patients' skin biopsies and the subsequent derivation of iPSCs using an efficient integrative-free mRNA-based somatic reprogramming approach.

86-OR: Self-Organized Insulin-Producing ß-Cells Differentiated from Human Omentum-Derived Stem Cells

Human omentum-derived mesenchymal stem cells (hO-MSCs) possess a great potential to differentiate into multiple lineages and have self-renewal capacities, allowing them to be utilized as patient-specific cell-based therapeutics. Although various stem cell-derived insulin-producing β-cells have been proposed for the treatment of diabetes mellitus, developing an efficient method to establish highly functionalized insulin-producing β-cells remains a challenging task. Here, we aimed to develop a novel cell culture platform that could regulate important cell-cell/cell-matrix interactions by introducing a fibroblast growth factor 2 (FGF2)-immobilized matrix that can support cell adhesion, proliferation, and differentiation of hO-MSCs into insulin-producing β-cells. Our initial findings demonstrated that cells cultured on an FGF2-immobilized matrix were able to self-organize into insulin-producing β-cell-like progenitors, as evident from the upregulation of pancreatic β-cell-specific markers such as PDX-1, Insulin, and Glut-2. Furthermore, heparan sulfate proteoglycan, gap junction proteins including Cx36 and Cx43, and cell adhesion molecules such as E-cadherin and Ncam1 were significantly upregulated, leading to cell maturation and insulin secretion in the pancreatic niche environment. These results suggest that our novel platform could promote β-cell differentiation and glucose-stimulated insulin secretion from hO-MSCs by enhancing cell-cell junction proteins such as connexins (Cx36, Cx43), E-cadherin, and N-cam. Such a cell culture platform can offer novel strategies to obtain functional pancreatic β-cells from a patient-specific cell sources, ultimately enabling better treatments for diabetes mellitus. Disclosure Y.Hwang: None. Funding National Research Foundation of Korea (2019R1A5A8083404)

Self-organized insulin-producing β-cells differentiated from human omentum-derived stem cells and their in vivo therapeutic potential

BackgroundHuman omentum-derived mesenchymal stem cells (hO-MSCs) possess great potential to differentiate into multiple lineages and have self-renewal capacity, allowing them to be utilized as patient-specific cell-based therapeutics. Although the use of various stem cell-derived β-cells has been proposed as a novel approach for treating diabetes mellitus, developing an efficient method to establish highly functional β-cells remains challenging.MethodsWe aimed to develop a novel cell culture platform that utilizes a fibroblast growth factor 2 (FGF2)-immobilized matrix to regulate the adhesion and differentiation of hO-MSCs into insulin-producing β-cells via cell–matrix/cell–cell interactions. In our study, we evaluated the in vitro differentiation potential of hO-MSCs cultured on an FGF2-immobilized matrix and a round-bottom plate (RBP). Further, the in vivo therapeutic efficacy of the β-cells transplanted into kidney capsules was evaluated using animal models with streptozotocin (STZ)-induced diabetes.ResultsOur findings demonstrated that cells cultured on an FGF2-immobilized matrix could self-organize into insulin-producing β-cell progenitors, as evident from the upregulation of pancreatic β-cell-specific markers (PDX-1, Insulin, and Glut-2). Moreover, we observed significant upregulation of heparan sulfate proteoglycan, gap junction proteins (Cx36 and Cx43), and cell adhesion molecules (E-cadherin and Ncam1) in cells cultured on the FGF2-immobilized matrix. In addition, in vivo transplantation of differentiated β-cells into animal models of STZ-induced diabetes revealed their survival and engraftment as well as glucose-sensitive production of insulin within the host microenvironment, at over 4 weeks after transplantation.ConclusionsOur findings suggest that the FGF2-immobilized matrix can support initial cell adhesion, maturation, and glucose-stimulated insulin secretion within the host microenvironment. Such a cell culture platform can offer novel strategies to obtain functional pancreatic β-cells from patient-specific cell sources, ultimately enabling better treatment for diabetes mellitus.Graphical

MiR-451a and let-7i-5p loaded extracellular vesicles attenuate heme-induced inflammation in hiPSC-derived endothelial cells.

Hemolysis is associated with many pathologies, including trauma, sepsis, hemorrhagic stroke, malaria, and genetic disorders such as sickle cell disease (SCD). When hemolysis occurs, free-heme drives vascular inflammation, resulting in oxidative tissue damage and cardiometabolic complications. A better understanding of heme clearance and detoxification is essential to preventing sustained tissue damage. Human induced pluripotent stem cell (hiPSC)-derived endothelial cells (hiPSC-ECs) provide a novel source of patient-specific cells and tissues for disease modeling, drug discovery, and regenerative therapeutics. Here we report the use of hiPSC-ECs to elucidate the role of miR-451a and let-7i-5p-loaded extracellular vesicles (EVs, such as exosomes) in the inflammatory response to free-heme as a model for heme-induced inflammation. We provide evidence of a significant correlation between miR-451a and let-7i-5p-loaded circulating exosomes in plasmodium-infected patients with reported clinical benchmarks of malaria-severity (e.g., Hemoglobin (Hb) levels, white blood cell counts). Additionally, we determined that exposure of Plasmodium falciparum (Pf) parasites to EVs, loaded with either miRNA, significantly reduces their counts in vitro. Using hiPSCs derived from individuals with wild-type Hb (HbAA) or homozygous sickle cell mutated Hb (HbSS) genotypes, we demonstrate that heme-treated hiPSC-ECs secreted inflammatory products (cytokines, chemokines and growth factors) into supporting media at concentrations that were similar to that reported in HbAA and HbSS serum. This inflammatory response was attenuated by exposure with miR-451a or let-7i-5p-loaded EVs. We also found a decrease in transcription of ICAM1 and P-Selectin, as well as the secretion of key inflammatory cytokines (e.g., CXCL10, TNF-α, and IFN-γ). Based on these findings, we propose a model in which increased levels of exosomal miR-451a and let-7i-5p in Plasmodium-infected individuals will attenuate inflammatory responses to free-heme and parasite-derived products. As a result, infected erythrocytes will less likely adhere to the endothelium, sequester in brain micro vessels, and reduce vaso-occlusive crises that exacerbate cerebral malaria.

Human induced pluripotent stem cells integrate, create synapses and extend long axons after spinal cord injury.

Numerous interventions have been explored in animal models using cells differentiated from human induced pluripotent stem cells (iPSCs) in the context of neural injury with some success. Our work seeks to transplant cells that are generated from hiPSCs into regionally specific spinal neural progenitor cells (sNPCs) utilizing a novel accelerated differentiation protocol designed for clinical translation. We chose a xenotransplantation model because our laboratory is focused on the behaviour of human cells in order to bring this potential therapy to translation. Cells were transplanted into adult immunodeficient rats after moderate contusion spinal cord injury (SCI). Twelve weeks later, cells derived from the transplanted sNPCs survived and differentiated into neurons and glia that filled the lesion cavity and produced a thoracic spinal cord transcriptional program in vivo. Furthermore, neurogenesis and ionic channel expression were promoted within the adjacent host spinal cord tissue. Transplanted cells displayed robust integration properties including synapse formation and myelination by host oligodendrocytes. Axons from transplanted hiPSC sNPC‐derived cells extended both rostrally and caudally from the SCI transplant site, rostrally approximately 6 cm into supraspinal structures. Thus, iPSC‐derived sNPCs may provide a patient‐specific cell source for patients with SCI that could provide a relay system across the site of injury.

High-Throughput Culture Method of Induced Pluripotent Stem Cell Derived Alveolar Epithelial Cells Using Floating Matrigel Droplets

<h3>Purpose</h3> Induced pluripotent stem cell derived alveolar epithelial cells (iPSC-AECs) are a patient specific cell source for bio-engineering of human pulmonary epithelium. Disease modeling and therapeutic applications require cost effective and technically feasible differentiation and expansion protocols. Current culture protocols are labor and resource intensive and not scalable for large animal or human studies. We developed a scalable culture protocol switching from adherent hydrogel drops to suspension culture. <h3>Methods</h3> Genetically modified iPSCs with fluorescent reporters for Nkx2.1 and SPC underwent differentiation in a stepwise culture protocol that mimics lung development. Cells were purified, sorted and embedded in a liquid Matrigel precursor. Homogenous liquid precursor was added to culture plates to form adherent droplets or to a silicone mold to form cell laden Matrigel spheroids which were subsequently transferred to spinner flasks with media as floating droplets. After culture, monolayer spheres of iPSC-AECs were digested and trypsinized to form single cell suspensions. Equal numbers of iPSC-AECs from the two culture conditions were seeded into decellularized lung scaffolds. <h3>Results</h3> iPSC-AECs cultured in floating droplets were more proliferative than adherent droplets, with significantly higher total cell counts and Ki67 expression. Equivalent expression of the distal lung markers Nkx2.1, SPC, and AQP5 was observed for both culture conditions in isolated iPSC-AECs and iPSC-AECs from recellularized lung scaffolds. Similar histologic appearance was appreciated on lungs recellularized from both culture groups. The floating droplet method was more time and cost efficient. <h3>Conclusion</h3> We report a simple, scalable, new culture method for iPSC-AECs with stable cellular phenotype that could support large animal and human studies. Our protocol enhanced proliferation and reduced labor requirements compared to previously reported methods.

A High-Efficiency Method for the Production of Endothelial Cells from Human Induced Pluripotent Stem Cells.

Endothelial cells (ECs) are important components of the circulatory system. These cells can be used for in vitro modeling of cardiovascular diseases and in regenerative medicine to promote vascularization of engineered tissue constructs. However, low proliferative capacity and patient-to-patient variability limit the use of primary ECs in the clinic and disease modeling. ECs differentiated from human induced pluripotent stem cells (iPSCs) can serve as a viable alternative to primary ECs for these applications. This is because human iPSCs can proliferate indefinitely and have the potential to differentiate into a variety of somatic cell lines, providing a renewable source of patient-specific cells. Here, we present an optimized, highly reproducible method for the differentiation of human iPSCs toward vascular ECs. The protocol relies on the activation of the WNT signaling pathway and the use of growth factors and small molecules. The resulting iPSC-derived ECs can be cultured for multiple passages without losing their functionality and are suitable for both in vitro and in vivo studies.

The collagen scaffold supports hiPSC-derived NSC growth and restricts hiPSC.

The human induced pluripotent stem cells (hiPSC) are one of the promising candidates as patient specific cell source for autologous transplantation or modeling of diseases. The collagen (Col) scaffolds have been shown suitable to create in vitro biomimetic microenvironment for human neural stem cells, but their ability to accommodate stem cells at different stages of neural differentiation has not been verified yet. In this paper we compare lineage related hiPSC during neural differentiation for their ability to colonize Col scaffold. We have also focused on modification of collagen physicochemical properties with improved mechanical and thermal stability, without loss of its biological activity. The hiPSC expressing markers of pluripotency (OCT4, SOX2, NANOG) after neural commitment are NESTIN, GFAP, PDGFR alpha, beta- TUBULIN III, MAP-2, DCX, GalC positive. We have shown, that Col scaffold was not preferable for hiPSC culture, while the neurally committed population after seeding on Col scaffolds revealed good adhesion, viability, proliferation, along with sustaining markers of neuronal and glial differentiation. The Col scaffold-based 3D culture of hiPSC-NSCs may serve as a research tool for further translational studies.

Striking parallels between carotid body glomus cell and adrenal chromaffin cell development

Carotid body glomus cells mediate essential reflex responses to arterial blood hypoxia. They are dopaminergic and secrete growth factors that support dopaminergic neurons, making the carotid body a potential source of patient-specific cells for Parkinson's disease therapy. Like adrenal chromaffin cells, which are also hypoxia-sensitive, glomus cells are neural crest-derived and require the transcription factors Ascl1 and Phox2b; otherwise, their development is little understood at the molecular level. Here, analysis in chicken and mouse reveals further striking molecular parallels, though also some differences, between glomus and adrenal chromaffin cell development. Moreover, histology has long suggested that glomus cell precursors are ‘émigrés’ from neighbouring ganglia/nerves, while multipotent nerve-associated glial cells are now known to make a significant contribution to the adrenal chromaffin cell population in the mouse. We present conditional genetic lineage-tracing data from mice supporting the hypothesis that progenitors expressing the glial marker proteolipid protein 1, presumably located in adjacent ganglia/nerves, also contribute to glomus cells. Finally, we resolve a paradox for the ‘émigré’ hypothesis in the chicken - where the nearest ganglion to the carotid body is the nodose, in which the satellite glia are neural crest-derived, but the neurons are almost entirely placode-derived - by fate-mapping putative nodose neuronal 'émigrés' to the neural crest.

Human iPSC-derived chondrocytes mimic juvenile chondrocyte function for the dual advantage of increased proliferation and resistance to IL-1\u03b2

BackgroundInduced pluripotent stem cells (iPSC) provide an unlimited patient-specific cell source for regenerative medicine. Adult cells have had limited success in cartilage repair, but juvenile chondrocytes (from donors younger than 13 years of age) have been identified to generate superior cartilage. With this perspective, the aim of these studies was to compare the human iPSC-derived chondrocytes (hiChondrocytes) to adult and juvenile chondrocytes and identify common molecular factors that govern their function.MethodsPhenotypic and functional characteristics of hiChondrocytes were compared to juvenile and adult chondrocytes. Analyses of global gene expression profiling, independent gene expression, and loss-of-function studies were utilized to test molecular factors having a regulatory effect on hiChondrocytes and juvenile chondrocyte function.ResultsHere, we report that the iPSC-derived chondrocytes mimic juvenile chondrocytes in faster cell proliferation and resistance to IL-1β compared to adult chondrocytes. Whole genome transcriptome analyses revealed unique ECM factors and immune response pathways to be enriched in both juvenile and iPSC-derived chondrocytes as compared to adult chondrocytes. Loss-of-function studies demonstrated that CD24, a cell surface receptor enriched in both juvenile chondrocytes and hiChondrocytes, is a regulatory factor in both faster proliferation and resistance to proinflammatory cues in these chondrocyte populations.ConclusionsOur studies identify that hiChondrocytes mimic juvenile chondrocytes for the dual advantage of faster proliferation and a reduced response to the inflammatory cytokine IL-1β. While developmental immaturity of iPSC-derived cells can be a challenge for tissues like muscle and brain, our studies demonstrate that it is advantageous for a tissue like cartilage that has limited regenerative ability in adulthood.

Understanding neurodevelopmental disorders using human pluripotent stem cell-derived neurons.

Research into psychiatric disorders has long been hindered by the lack of appropriate models. Induced pluripotent stem cells (iPSCs) offer an unlimited source of patient-specific cells, which in principle can be differentiated into all disease-relevant somatic cell types to create in vitro models of the disorder of interest. Here, neuronal differentiation protocols available for this purpose and the current progress on iPSCs-based models of schizophrenia, autism spectrum disorders and bipolar disorder were reviewed. We also discuss the impact of the recently developed CRISPR/Cas9 genome editing tool in the disease modeling field. Genetically engineered mutation of disease risk alleles in well characterized reference "control" hPSCs or correction of disease risk variants in patient iPSCs has been used as a powerful means to establish causality of the identified cellular pathology. Together, iPSC reprogramming and CRISPR/CAS9 genome editing technology have already significantly contributed to our understanding of the developmental origin of some major psychiatric disorders. The challenge ahead is the identification of shared mechanisms in their etiology, which will ultimately be relevant to the development of new treatments.

Endothelial and smooth muscle cells derived from human cardiac explants demonstrate angiogenic potential and suitable for design of cell-containing vascular grafts

BackgroundEndothelial and smooth muscle cells are considered promising resources for regenerative medicine and cell replacement therapy. It has been shown that both types of cells are heterogeneous depending on the type of vessels and organs in which they are located. Therefore, isolation of endothelial and smooth muscle cells from tissues relevant to the area of research is necessary for the adequate study of specific pathologies. However, sources of specialized human endothelial and smooth muscle cells are limited, and the search for new sources is still relevant. The main goal of our study is to demonstrate that functional endothelial and smooth muscle cells can be obtained from an available source—post-surgically discarded cardiac tissue from the right atrial appendage and right ventricular myocardium.MethodsHeterogeneous primary cell cultures were enzymatically isolated from cardiac explants and then grown in specific endothelial and smooth muscle growth media on collagen IV-coated surfaces. The population of endothelial cells was further enriched by immunomagnetic sorting for CD31, and the culture thus obtained was characterized by immunocytochemistry, ultrastructural analysis and in vitro functional tests. The angiogenic potency of the cells was examined by injecting them, along with Matrigel, into immunodeficient mice. Cells were also seeded on characterized polycaprolactone/chitosan membranes with subsequent analysis of cell proliferation and function.ResultsEndothelial cells isolated from cardiac explants expressed CD31, VE-cadherin and VEGFR2 and showed typical properties, namely, cytoplasmic Weibel-Palade bodies, metabolism of acetylated low-density lipoproteins, formation of capillary-like structures in Matrigel, and production of extracellular matrix and angiogenic cytokines. Isolated smooth muscle cells expressed extracellular matrix components as well as α-actin and myosin heavy chain. Vascular cells derived from cardiac explants demonstrated the ability to stimulate angiogenesis in vivo. Endothelial cells proliferated most effectively on membranes made of polycaprolactone and chitosan blended in a 25:75 ratio, neutralized by a mixture of alkaline and ethanol. Endothelial and smooth muscle cells retained their functional properties when seeded on the blended membranes.ConclusionsWe established endothelial and smooth muscle cell cultures from human right atrial appendage and right ventricle post-operative explants. The isolated cells revealed angiogenic potential and may be a promising source of patient-specific cells for regenerative medicine.

Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids.

Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regenerative capacity of adult human hearts, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have received significant attention because they provide a patient specific cell source to regenerate damaged hearts. Despite the progress, current human hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. In this manuscript, we examined the effects of two important factors on the functions of nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. The results from these studies will allow for the development of suitable nanowired hiPSC cardiac spheroids to effectively deliver hiPSC-CMs for heart repair.

Human iPSC-derived osteoblasts and osteoclasts together promote bone regeneration in 3D biomaterials.

Bone substitutes can be designed to replicate physiological structure and function by creating a microenvironment that supports crosstalk between bone and immune cells found in the native tissue, specifically osteoblasts and osteoclasts. Human induced pluripotent stem cells (hiPSC) represent a powerful tool for bone regeneration because they are a source of patient-specific cells that can differentiate into all specialized cell types residing in bone. We show that osteoblasts and osteoclasts can be differentiated from hiPSC-mesenchymal stem cells and macrophages when co-cultured on hydroxyapatite-coated poly(lactic-co-glycolic acid)/poly(L-lactic acid) (HA–PLGA/PLLA) scaffolds. Both cell types seeded on the PLGA/PLLA especially with 5% w/v HA recapitulated the tissue remodeling process of human bone via coupling signals coordinating osteoblast and osteoclast activity and finely tuned expression of inflammatory molecules, resulting in accelerated in vitro bone formation. Following subcutaneous implantation in rodents, co-cultured hiPSC-MSC/-macrophage on such scaffolds showed mature bone-like tissue formation. These findings suggest the importance of coupling matrix remodeling through osteoblastic matrix deposition and osteoclastic tissue resorption and immunomodulation for tissue development.

Research progress on disease models and gene therapy of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked, recessive and lethal genetic disease, which usually caused by gene mutations and the underlying mechanisms are complicated and diverse. The causal gene of DMD is the largest one in human that locates in the region of Xp21.2, encoding dystrophin. Currently there is no effective treatment for DMD patients. The treatment of DMD depends on gene mutation and molecular mechanism study of the disease, which requires reliable disease models such as mdx mouse model. Recently, researchers have increasingly discovered gene therapy strategies for DMD, and the efficacy has been demonstrated in DMD animal models. In addition, induced pluripotent stem cell technology can provide patient-specific cell source, offering a new platform for mechanism and therapy study of DMD.

Stem cells: to be born great, achieve greatness, or have greatness thrust upon them?

Induced pluripotent stem cells (iPSCs) have opened new doors in providing an ethical, patient-specific cell source towards tissue engineering. Developing these therapies involves the production of reprogrammed iPSCs, expanding them while maintaining pluripotency, then differentiating them into functional tissues. To bring these therapies to the clinic, efficient and GMP-compliant manufacturing methods are required. In this Research Highlight, we describe recent innovations to several aspects of the pluripotent cell therapy pipeline.

Differentiation of Human Induced Pluripotent Stem Cells to Chondrocytes.

Human induced pluripotent stem (iPS) cells are relevant tools for modeling human skeletal development and disease, and represent a promising source of patient-specific cells for the regeneration of skeletal tissue, such as articular cartilage. Devising efficient and reproducible strategies, which closely mimic the physiological chondrogenic differentiation process, will be necessary to generate functional chondrocytes from human iPS cells. Our previous study demonstrated the generation of chondrogenically committed human iPS cells via the enrichment of a mesenchymal-like progenitor population, application of appropriate high-density culture conditions, and stimulation with bone morphogenetic protein-2 (Bmp-2). The differentiated iPS cells showed temporal expression of cartilage genes and the accumulation of a cartilaginous extracellular matrix in vitro. In this chapter, we provide detailed methodologies for the differentiation of human iPS cells to the chondrogenic lineage and describe protocols for the analysis of chondrogenic differentiation.

Modeling Diamond Blackfan Anemia in Vivo Using Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells has limited their use for modeling of hematological diseases. We previously reported a strategy to respecify lineage-restricted CD34+hematopoietic precursors derived from iPSCs into multilineage progenitors that can be expanded in vitro and transplanted in vivo. Five transcription factors, HOXA9, ERG, RORA, SOX4 and MYB, enable expansion and maintenance of primitive CD34+CD38- cells, and allow their differentiation upon transgene silencing. Respecified iPSC-derived hematopoietic progenitors give rise to robust short-term engraftment with myeloid and erythroid lineages. Notably, the erythrocytes undergo definitive maturation and hemoglobin switching to express β-globin in vivo. This system presents a useful platform for modeling hematological disorders due to its capacity to generate large numbers of engraftable disease cells for in vitro and in vivo screens.Congenital anemias, such as Diamond Blackfan anemia (DBA) represent a defined system for understanding red blood cell development and more common idiopathic anemias. To model this disease in vitro, we combined factor-induced respecification with stepwise erythroid maturation. We show that respecified iPSCs from DBA patients recapitulate the defect in erythroid differentiation. Consistent with clinical observations, early erythroblasts show impaired proliferation marked by increased apoptosis and p21 expression, while terminal maturation is unaffected. Interestingly, while early passage iPSC lines display a profound erythroid defect, continuous passage restores a near-normal capacity for erythroid differentiation. Furthermore, transplanted DBA iPSC-derived progenitors give rise to normal myeloid, but fail to generate erythroid, engraftment. This validates this system as a powerful tool for disease modeling and drug discovery. Mice transplanted with DBA iPSC-derived progenitors treated with conventional anemia drugs, such as dexamethasone, show a modest improvement in human erythroid output suggesting the need for novel treatments. Using high-throughput chemical screens, we have identified several signaling pathways which may be attractive therapeutic targets in DBA and other anemias. Further characterization is currently underway to better target defective erythropoiesis and dissect underlying disease mechanisms. DisclosuresZon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Disease modeling and cell based therapy with iPSC: future therapeutic option with fast and safe application.

Induced pluripotent stem cell (iPSC) technology has shown us great hope to treat various human diseases which have been known as untreatable and further endows personalized medicine for future therapy without ethical issues and immunological rejection which embryonic stem cell (hES) treatment has faced. It has been agreed that iPSCs knowledge can be harnessed from disease modeling which mimics human pathological development rather than trials utilizing conventional rodent and cell lines. Now, we can routinely generate iPSC from patient specific cell sources, such as skin fibroblast, hair follicle cells, patient blood samples and even urine containing small amount of epithelial cells. iPSC has both similarity and dissimilarity to hES. iPSC is similar enough to regenerate tissue and even full organism as ES does, however what we want for therapeutic advantage is limited to regenerated tissue and lineage specific differentiation. Depending on the lineage and type of cells, both tissue memory containing (DNA rearrangement/epigenetics) and non-containing iPSC can be generated. This makes iPSC even better choice to perform disease modeling as well as cell based therapy. Tissue memory containing iPSC from mature leukocytes would be beneficial for curing cancer and infectious disease. In this review, the benefit of iPSC for translational approaches will be presented.

Induction of Multipotential Hematopoietic Progenitors from Human Pluripotent Stem Cells via Respecification of Lineage-Restricted Precursors

Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells (HSPCs) has limited their characterization to invitro assays. We report a strategy to respecify lineage-restricted CD34(+)CD45(+) myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded invitro and engrafted invivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential invitro and maintained primitive CD34(+)CD38(-) cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, conferred engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching invivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription-factor-mediated engraftment of blood progenitors from human pluripotent cells.