Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

Transfusion of red blood cells (RBCs) is a life-saving intervention for anemic patients. Human induced pluripotent stem cells (iPSC) have the capability to expand and differentiate into RBCs (iPSC-RBCs). Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. iPSC lines were produced from human peripheral blood mononuclear cells by transient expression of plasmids containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes. Human iPSC-RBCs were generated in culture supplemented with human platelet lysate, and were CD34- CD235a+ CD233+ CD49dlow CD71low ; about 13% of iPSC-RBCs were enucleated before transfusion. Systemic administration of clodronate liposomes (CL) and cobra venom factor (CVF) to NOD scid gamma (NSG) mice markedly promoted the circulatory survival of human iPSC-RBCs following transfusion. While iPSC-RBCs progressively decreased with time, 90% of circulating iPSC-RBCs were enucleated 1 day after transfusion (CD235a+ CD233+ CD49d- CD71- ). Surprisingly, human iPSC-RBCs reappeared in the peripheral circulation at 3 weeks after transfusion at levels more than 8-fold higher than at 1 h after transfusion. Moreover, a substantial portion of the transfused nucleated iPSC-RBCs preferentially homed to the bone marrow, and were detectable at 24 days after transfusion. These results suggest that nucleated human iPSC-derived cells that homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into peripheral blood. The results offer a new model using human peripheral blood-derived iPSC and CL/CVF-treated NSG mice to investigate the development and circulation of human erythroid cells in vivo.

BMI1 enables extensive expansion of functional erythroblasts from human peripheral blood mononuclear cells.

Transfusion of red blood cells (RBCs) from ABO-matched but genetically unrelated donors is commonly used for treating anemia and acute blood loss. Increasing demand and insufficient supply for donor RBCs, especially those of universal blood types or free of known and unknown pathogens, has called for ex vivo generation of functional RBCs by large-scale cell culture. However, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex vivo remains challenging, due to our inability to either extensively expand primary RBC precursors (erythroblasts) or achieve efficient enucleation once erythroblasts have been expanded and induced to differentiation and maturation. Here, we report that ectopic expression of the human BMI1 gene confers extensive expansion of human erythroblasts, which can be derived readily from adult peripheral blood mononuclear cells of either healthy donors or sickle cell patients. These extensively expanded erythroblasts (E3s) are able to proliferate exponentially (>1 trillion-fold in 2 months) in a defined culture medium. Expanded E3 cells are karyotypically normal and capable of terminal maturation with approximately 50% enucleation. Additionally, E3-derived cRBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Therefore, we provide a facile approach of generating physiological numbers of human functional erythroblasts ex vivo.

PDGFRα marks distinct perivascular populations with different osteogenic potential within adipose tissue.

The perivascular niche within adipose tissue is known to house multipotent cells, including osteoblast precursors. However, the identity of perivascular subpopulations that may mineralize or ossify most readily is not known. Here, we utilize inducible PDGFRα (platelet-derived growth factor alpha) reporter animals to identify subpopulations of perivascular progenitor cells. Results showed that PDGFRα-expressing cells are present in four histologic niches within inguinal fat, including two perivascular locations. PDGFRα+ cells are most frequent within the tunica adventitia of arteries and veins, where PDGFRα+ cells populate the inner aspects of the adventitial layer. Although both PDGFRα+ and PDGFRα- fractions are multipotent progenitor cells, adipose tissue-derived PDGFRα+ stromal cells proliferate faster and mineralize to a greater degree than their PDGFRα- counterparts. Likewise, PDGFRα+ ectopic implants reconstitute the perivascular niche and ossify to a greater degree than PDGFRα- cell fractions. Adventicytes can be further grouped into three distinct groups based on expression of PDGFRα and/or CD34. When further partitioned, adventicytes co-expressing PDGFRα and CD34 represented a cell fraction with the highest mineralization potential. Long-term tracing studies showed that PDGFRα-expressing adventicytes give rise to adipocytes, but not to other cells within the vessel wall under homeostatic conditions. However, upon bone morphogenetic protein 2 (BMP2)-induced ossicle formation, descendants of PDGFRα+ cells gave rise to osteoblasts, adipocytes, and "pericyte-like" cells within the ossicle. In sum, PDGFRα marks distinct perivascular osteoprogenitor cell subpopulations within adipose tissue. The identification of perivascular osteoprogenitors may contribute to our improved understanding of pathologic mineralization/ossification.

Human NOTCH4 is a key target of RUNX1 in megakaryocytic differentiation.

Megakaryocytes (MKs) in adult marrow produce platelets that play important roles in blood coagulation and hemostasis. Monoallelic mutations of the master transcription factor gene RUNX1 lead to familial platelet disorder (FPD) characterized by defective MK and platelet development. However, the molecular mechanisms of FPD remain unclear. Previously, we generated human induced pluripotent stem cells (iPSCs) from patients with FPD containing a RUNX1 nonsense mutation. Production of MKs from the FPD-iPSCs was reduced, and targeted correction of the RUNX1 mutation restored MK production. In this study, we used isogenic pairs of FPD-iPSCs and the MK differentiation system to identify RUNX1 target genes. Using integrative genomic analysis of hematopoietic progenitor cells generated from FPD-iPSCs, and mutation-corrected isogenic controls, we identified 2 gene sets the transcription of which is either up- or downregulated by RUNX1 in mutation-corrected iPSCs. Notably, NOTCH4 expression was negatively controlled by RUNX1 via a novel regulatory DNA element within the locus, and we examined its involvement in MK generation. Specific inactivation of NOTCH4 by an improved CRISPR-Cas9 system in human iPSCs enhanced megakaryopoiesis. Moreover, small molecules known to inhibit Notch signaling promoted MK generation from both normal human iPSCs and postnatal CD34+ hematopoietic stem and progenitor cells. Our study newly identified NOTCH4 as a RUNX1 target gene and revealed a previously unappreciated role of NOTCH4 signaling in promoting human megakaryopoiesis. Our work suggests that human iPSCs with monogenic mutations have the potential to serve as an invaluable resource for discovery of novel druggable targets.

Targeted correction of RUNX1 mutation in FPD patient-specific induced pluripotent stem cells rescues megakaryopoietic defects.

Familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease of the hematopoietic system that is caused by heterozygous mutations in RUNX1. FPD/AML patients have a bleeding disorder characterized by thrombocytopenia with reduced platelet numbers and functions, and a tendency to develop AML. No suitable animal models exist for FPD/AML, as Runx11/2 mice and zebra fish do not develop bleeding disorders or leukemia. Here we derived induced pluripotent stem cells (iPSCs) from 2 patients in a family with FPD/AML, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in 1 FPD iPSC line through gene targeting, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients.

Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation.

Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult ß-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa ß-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479.

Deletion of proapoptotic Puma selectively protects hematopoietic stem and progenitor cells against high-dose radiation.

Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted, but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here, we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell-autonomous manner. Unexpectedly, loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly, null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation, thereby accelerating hematopoietic regeneration. Consistent with these findings, Puma is required for radiation-induced apoptosis in HSCs and HPCs, and Puma is selectively induced by irradiation in primitive hematopoietic cells, and this induction is impaired in Puma-heterozygous cells. Together, our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy.

The role of SDF-1α/Rac pathway in the regulation of endothelial progenitor cell polarity; homing and expression of Rac1, Rac2 during endothelial repair.

The Stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) system is considered to be of great importance in diseases involving cardiogenesis and angiogenesis. The SDF-1α-RAC signaling pathway plays a pivotal role in a cell's polarity and serves to activate cell morphology variation and to control the direction of migration. We aimed to study whether the polarity of endothelial progenitor cells (EPCs) is changed by the induction of the SDF-1α-RAC signaling pathway, to investigate the mechanism of the effect of polarity on the homing action of EPCs, and to explore the gene and protein expression of Rac1/2 during endothelial repair. We measured the EPC characteristics of polarity induced by various final concentrations of SDF-1α; our observations included morphology variation, migration direction, and excursion. Of the dynamic variation and cytoskeleton rearrangement of EPCs induced by different final concentrations of SDF-1α, the most obvious variation was exhibited at the SDF-1α concentration of 200 ng/ml. Obvious polarity variations were also found in the EPCs and signal receptors induced by the SDF-1α concentration of 200 ng/ml. The Western blot analysis of Rac1 and Rac2 showed that the addition of AMD 3100 significantly inhibited the expression of Rac. The SDF-1α pathway potentially regulates the expression of Rac1/2. The actual excursion vector and the direction of the migration of EPCs induced by SDF-1α follows polarity, thus indicating the importance of further exploration regarding the homing induction of EPCs.

BMP4 regulates vascular progenitor development in human embryonic stem cells through a Smad-dependent pathway.

The signals that direct pluripotent stem cell differentiation into lineage-specific cells remain largely unknown. Here, we investigated the roles of BMP on vascular progenitor development from human embryonic stem cells (hESCs). In a serum-free condition, hESCs sequentially differentiated into CD34+CD31-, CD34+CD31+, and then CD34-CD31+ cells during vascular cell development. CD34+CD31+ cells contained vascular progenitor population that gives rise to endothelial cells and smooth muscle cells. BMP4 promoted hESC differentiation into CD34+CD31+ cells at an early stage. In contrast, TGFbeta suppressed BMP4-induced CD34+CD31+ cell development, and promoted CD34+CD31- cells that failed to give rise to either endothelial or smooth muscle cells. The BMP-Smad inhibitor, dorsomorphin, inhibited phosphorylation of Smad1/5/8, and blocked hESC differentiation to CD34+CD31+ progenitor cells, suggesting that BMP Smad-dependent signaling is critical for CD34+CD31+ vascular progenitor development. Our findings provide new insight into how pluripotent hESCs differentiate into vascular cells.

Endothelial cells regulate cardiomyocyte development from embryonic stem cells.

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac-specific genes, including alpha-MHC and MLC-2V, was significantly decreased in EphB4-null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac-specific genes were significantly enhanced by co-culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4-deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes.

iPSCs from people with MS can differentiate into oligodendrocytes in a homeostatic but not an inflammatory milieu.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS) that results in variable severities of neurodegeneration. The understanding of MS has been limited by the inaccessibility of the affected cells and the lengthy timeframe of disease development. However, recent advances in stem cell technology have facilitated the bypassing of some of these challenges. Towards gaining a greater understanding of the innate potential of stem cells from people with varying degrees of disability, we generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells derived from stable and progressive MS patients, and then further differentiated them into oligodendrocyte (OL) lineage cells. We analyzed differentiation under both homeostatic and inflammatory conditions via sustained exposure to low-dose interferon gamma (IFNγ), a prominent cytokine in MS. We found that all iPSC lines differentiated into mature myelinating OLs, but chronic exposure to IFNγ dramatically inhibited differentiation in both MS groups, particularly if exposure was initiated during the pre-progenitor stage. Low-dose IFNγ was not toxic but led to an early upregulation of interferon response genes in OPCs followed by an apparent redirection in lineage commitment from OL to a neuron-like phenotype in a significant portion of the treated cells. Our results reveal that a chronic low-grade inflammatory environment may have profound effects on the efficacy of regenerative therapies.

A Neurotrophic Mechanism Directs Sensory Nerve Transit in Cranial Bone.

The flat bones of the skull are densely innervated during development, but little is known regarding their role during repair. We describe a neurotrophic mechanism that directs sensory nerve transit in the mouse calvaria. Patent cranial suture mesenchyme represents an NGF (nerve growth factor)-rich domain, in which sensory nerves transit. Experimental calvarial injury upregulates Ngf in an IL-1ß/TNF-α-rich defect niche, with consequent axonal ingrowth. In calvarial osteoblasts, IL-1ß and TNF-α stimulate Ngf and downstream NF-κB signaling. Locoregional deletion of Ngf delays defect site re-innervation and blunted repair. Genetic disruption of Ngf among LysM-expressing macrophages phenocopies these observations, whereas conditional knockout of Ngf among Pdgfra-expressing cells does not. Finally, inhibition of TrkA catalytic activity similarly delays re-innervation and repair. These results demonstrate an essential role of NGF-TrkA signaling in bone healing and implicate macrophage-derived NGF-induced ingrowth of skeletal sensory nerves as an important mediator of this repair.

Lysosomal protein surface expression discriminates fat- from bone-forming human mesenchymal precursor cells.

Tissue resident mesenchymal stem/stromal cells (MSCs) occupy perivascular spaces. Profiling human adipose perivascular mesenchyme with antibody arrays identified 16 novel surface antigens, including endolysosomal protein CD107a. Surface CD107a expression segregates MSCs into functionally distinct subsets. In culture, CD107alow cells demonstrate high colony formation, osteoprogenitor cell frequency, and osteogenic potential. Conversely, CD107ahigh cells include almost exclusively adipocyte progenitor cells. Accordingly, human CD107alow cells drove dramatic bone formation after intramuscular transplantation in mice, and induced spine fusion in rats, whereas CD107ahigh cells did not. CD107a protein trafficking to the cell surface is associated with exocytosis during early adipogenic differentiation. RNA sequencing also suggested that CD107alow cells are precursors of CD107ahigh cells. These results document the molecular and functional diversity of perivascular regenerative cells, and show that relocation to cell surface of a lysosomal protein marks the transition from osteo- to adipogenic potential in native human MSCs, a population of substantial therapeutic interest.

Comparison of skeletal and soft tissue pericytes identifies CXCR4+ bone forming mural cells in human tissues.

Human osteogenic progenitors are not precisely defined, being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells (MSCs). Notably, select human pericytes can develop into bone-forming osteoblasts. Here, we sought to define the differentiation potential of CD146+ human pericytes from skeletal and soft tissue sources, with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte. CD146+CD31-CD45- pericytes were derived by fluorescence-activated cell sorting from human periosteum, adipose, or dermal tissue. Periosteal CD146+CD31-CD45- cells retained canonical features of pericytes/MSC. Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo, while soft tissue pericytes did not readily. Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts, and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes. Conversely, enrichment of CXCR4+ pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell. In sum, human skeletal and soft tissue pericytes differ in their basal abilities to form bone. Diversity exists in soft tissue pericytes, however, and CXCR4+ pericytes represent an osteoblastogenic, non-adipocytic cell precursor. Indeed, enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering.

LKB1 suppresses androgen synthesis in a mouse model of hyperandrogenism via IGF-1 signaling.

Polycystic ovary syndrome (PCOS) is a major cause of anovulatory sterility in women, and most PCOS patients exhibit hyperandrogenism (HA). Liver kinase b1 (LKB1) is a tumor suppressor that has recently been reported to be involved in PCOS. However, the mechanism by which LKB1 affects HA has not previously been elucidated. We report here that ovarian LKB1 levels are significantly decreased in a female mouse model of HA. Moreover, we report that LKB1 expression is inhibited by elevated androgens via activation of androgen receptors. In addition, LKB1 treatment was observed to suppress androgen synthesis in theca cells and promote estrogen production in granulosa cells by regulating steroidogenic enzyme expression. As expected, LKB1 knockdown inhibited estrogen levels and enhanced androgen levels, and LKB1-transgenic mice were protected against HA. The effect of LKB1 appears to be mediated via IGF-1 signaling. In summary, we describe here a key role for LKB1 in controlling sex hormone levels.

The transcription factor Slug represses p16Ink4a and regulates murine muscle stem cell aging.

Activation of the p16Ink4a-associated senescence pathway during aging breaks muscle homeostasis and causes degenerative muscle disease by irreversibly dampening satellite cell (SC) self-renewal capacity. Here, we report that the zinc-finger transcription factor Slug is highly expressed in quiescent SCs of mice and functions as a direct transcriptional repressor of p16Ink4a. Loss of Slug promotes derepression of p16Ink4a in SCs and accelerates the entry of SCs into a fully senescent state upon damage-induced stress. p16Ink4a depletion partially rescues defects in Slug-deficient SCs. Furthermore, reduced Slug expression is accompanied by p16Ink4a accumulation in aged SCs. Slug overexpression ameliorates aged muscle regeneration by enhancing SC self-renewal through active repression of p16Ink4a transcription. Our results identify a cell-autonomous mechanism underlying functional defects of SCs at advanced age. As p16Ink4a dysregulation is the chief cause for regenerative defects of human geriatric SCs, these findings highlight Slug as a potential therapeutic target for aging-associated degenerative muscle disease.

Human perivascular stem cell-derived extracellular vesicles mediate bone repair.

The vascular wall is a source of progenitor cells that are able to induce skeletal repair, primarily by paracrine mechanisms. Here, the paracrine role of extracellular vesicles (EVs) in bone healing was investigated. First, purified human perivascular stem cells (PSCs) were observed to induce mitogenic, pro-migratory, and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture via elaboration of EVs. PSC-derived EVs shared mitogenic, pro-migratory, and pro-osteogenic properties of their parent cell. PSC-EV effects were dependent on surface-associated tetraspanins, as demonstrated by EV trypsinization, or neutralizing antibodies for CD9 or CD81. Moreover, shRNA knockdown in recipient cells demonstrated requirement for the CD9/CD81 binding partners IGSF8 and PTGFRN for EV bioactivity. Finally, PSC-EVs stimulated bone repair, and did so via stimulation of skeletal cell proliferation, migration, and osteodifferentiation. In sum, PSC-EVs mediate the same tissue repair effects of perivascular stem cells, and represent an 'off-the-shelf' alternative for bone tissue regeneration.

Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.

Precise genome editing in human induced pluripotent stem cells (iPSCs) significantly enhances our capability to use human iPSCs for disease modeling, drug testing and screening as well as investigation of human cell biology. In this study, we seek to achieve conditional expression of the CD55 gene in order to interrogate its functions. We used two human iPSC lines that have unique genotypes, and constructed an inducible Cas9 gene expression system that is integrated at the AAVS1 safe harbor site in the human genome. Using paired guide RNAs, we observed efficient knock-out with an intended deletion in the coding region of several genes including CD55 and ETV6 genes. This paired guide RNA approach enabled us to efficiently identify homozygous iPSC clones with an intended deletion. Once an iPSC clone lacking CD55 expression was identified and characterized, we were able to use the same doxycycline system to induce expression of a CD55 transgene from a piggyBac vector, in both undifferentiated and differentiated iPSCs. This single cell line of gene knock-out complemented with an inducible transgene is sufficient to achieve conditional expression of the CD55 gene. The methodology described here is broadly applicable to other genes in order to interrogate their functions.

A Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta-Thalassemia and Sickle Cell Disease.

Beta-thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with ß-thalassemia (ß-thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site-specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence-specific nuclease to enhance correction of a specific HBB mutation by homology-directed repair (HDR) is becoming straightforward, targeting various HBB mutations of ß-thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two ß-thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in ß-thal and SCD. Stem Cells Translational Medicine 2018;7:87-97.

Suppression of Transforming Growth Factor-ß Signaling Delays Cellular Senescence and Preserves the Function of Endothelial Cells Derived from Human Pluripotent Stem Cells.

Transplantation of vascular cells derived from human pluripotent stem cells (hPSCs) offers an attractive noninvasive method for repairing the ischemic tissues and for preventing the progression of vascular diseases. Here, we found that in a serum-free condition, the proliferation rate of hPSC-derived endothelial cells is quickly decreased, accompanied with an increased cellular senescence, resulting in impaired gene expression of endothelial nitric oxide synthase (eNOS) and impaired vessel forming capability in vitro and in vivo. To overcome the limited expansion of hPSC-derived endothelial cells, we screened small molecules for specific signaling pathways and found that inhibition of transforming growth factor-ß (TGF-ß) signaling significantly retarded cellular senescence and increased a proliferative index of hPSC-derived endothelial cells. Inhibition of TGF-ß signaling extended the life span of hPSC-derived endothelial and improved endothelial functions, including vascular network formation on Matrigel, acetylated low-density lipoprotein uptake, and eNOS expression. Exogenous transforming growth factor-ß1 increased the gene expression of cyclin-dependent kinase inhibitors, p15Ink4b , p16Ink4a , and p21CIP1 , in endothelial cells. Conversely, inhibition of TGF-ß reduced the gene expression of p15Ink4b , p16Ink4a , and p21CIP1 . Our findings demonstrate that the senescence of newly generated endothelial cells from hPSCs is mediated by TGF-ß signaling, and manipulation of TGF-ß signaling offers a potential target to prevent vascular aging. Stem Cells Translational Medicine 2017;6:589-600.