There is an ongoing need for innovative cell and regenerative medicine therapies to promote vascular repair and regeneration to meet the growing global health concern of ischemic cardiovascular diseases. Direct reprogramming of somatic cells into an induced pluripotent state caused scientists to reconsider prior theories of cell differentiation and develop approaches to directly reprogram cells in vivo into other states or fates to adaptively replace cells lost to aging or disease. While direct in vivo reprogramming of fibroblast to cardiomyocytes, neurons, or pancreatic beta islet cell states has been studied since 2008, direct in vivo reprogramming of fibroblasts into induced vasculogenic cells was not reported until 2017. This review provides a brief overview of the field of in vitro direct reprogramming of fibroblasts into induced endothelial cells over the past decade and identifies key similarities and differences in approaches. The primary focus of this review is to discuss in detail 4 published reports of direct in vivo reprogramming of fibroblasts into vasculogenic cell states and identify a series of questions for future studies that will clarify the reprogrammed cells and potential for using these approaches for translation into larger preclinical models or human subjects.

Diabetic wounds are complicated by underlying peripheral vasculopathy. Reliance on vascular endothelial growth factor (VEGF) therapy to improve perfusion makes logical sense, yet clinical study outcomes on rescuing diabetic wound vascularization have yielded disappointing results. Our previous work has identified that low endothelial phospholipase Cγ2 (PLCγ2) expression hinders the therapeutic effect of VEGF on the diabetic ischemic limb. In this work, guided by single-cell RNA sequencing of human wound edge, we test the efficacy of gene-targeted therapeutic demethylation intending to improve VEGF-mediated neovascularization. PLCγ2 expression was diminished in all five identified diabetic wound-edge endothelial subclusters encompassing arterial, venous, and capillary cells. Such low expression was associated with hypermethylated PLCγ2 promoter. PLCγ2 promoter was also hypermethylated at murine diabetic ischemic wound edge. To specifically demethylate endothelial PLCγ2 promoter during VEGF therapy, a CRISPR-dCas9-based demethylation cocktail was delivered to the ischemic wound edge using tissue nanotransfection (TNT) technology. Demethylation-based upregulation of PLCγ2 during VEGF therapy improved wound tissue blood flow with an increased abundance of von Willebrand factor (vWF)+/PLCγ2+ vascular tissue elements by activating p44/p42-mitogen-activated protein kinase (MAPK) → hypoxia-inducible factor [HIF]-1α pathway. Taken together, TNT-based delivery of plasmids to demethylate the PLCγ2 gene promoter activity led to significant improvements in VEGF therapy for cutaneous diabetic wounds, resulting in better perfusion and accelerated wound closure.

Purpose: To investigate the therapeutic potential of inducible pluripotent stem cell (hiPSC)-based vascular repair, we evaluated two vascular reparative cell populations, CD34+ cells derived from hiPSC (hiPSC-CD34+) and endothelial colony forming cells (ECFCs) derived from hiPSC (iPS-ECFCs), alone and in combination, in a type 2 diabetic (db/db) mouse model of DR. Methods: hiPSC-CD34+ cells (1 × 104) or iPSC- ECFCs (1 × 105) alone or in combination (1.1 × 105) were injected into the vitreous of immunosuppressed db/db mice with six months of established diabetes. One month post-injection, mice underwent electroretinography (ERG) and optical coherence tomography (OCT) to evaluate functional and structural retinal recovery with iPSC administration. Immunohistochemistry (IHC) was used to assess recruitment and incorporation of cells into the retinal vasculature. Retinas from the experimental groups were analyzed using Functional Proteomics via Reverse Phase Protein Array (RPPA). Results: Functional assessment via ERG demonstrated significant improvements in retinal response in the diabetic cohorts treated with either hiPSC-derived CD34+ cells or hiPSC-ECFCs. Retinal thickness, assessed by OCT, was restored to near-nondiabetic levels in mice treated with hiPSC-CD34+ cells alone and the combination group, whereas hiPSC-ECFCs alone did not significantly affect retinal thickness. One month following intravitreal injection, hiPSC-CD34+ cells were localized to perivascular regions, whereas hiPSC-ECFCs were observed to integrate directly into the retinal vasculature. RPPA analysis revealed interaction-significant changes, and this was interpreted as a combination-specific, non-additive host responses (m6A, PI3K–AKT–mTOR, glycolysis, endothelial junction pathways). Conclusions: The studies support that injection of hiPSC-CD34+ cells and hiPSC-ECFCs, both individually and in combination, showed benefit; however, iPSC combination-specific effects were identified by measurement of retinal thickness and by RPPA.

Achieving bone union remains a significant clinical dilemma. The use of osteoinductive agents, specifically bone morphogenetic proteins (BMPs), has gained wide attention. However, multiple side effects, including increased incidence of cancer, have renewed interest in investigating alternatives that provide safer, yet effective bone regeneration. Here we demonstrate the robust bone healing capabilities of the main megakaryocyte (MK) growth factor, thrombopoietin (TPO), and second-generation TPO agents using multiple animal models, including mice, rats, and pigs. This bone healing activity is shown in two fracture models (critical-sized defect [CSD] and closed fracture) and with local or systemic administration. Our transcriptomic analyses, cellular studies, and protein arrays demonstrate that TPO enhances multiple cellular processes important to fracture healing, particularly angiogenesis, which is required for bone union. Finally, the therapeutic potential of thrombopoietic agents is high since they are used in the clinic for other indications (eg, thrombocytopenia) with established safety profiles and act upon a narrowly defined population of cells.

BACKGROUND:Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive.METHODS:We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of Abcg2-expressing ECs to vessel development and regeneration, we developed Abcg2CreErt2;ROSA TdTomato mice and performed lineage tracing during mouse development and during tissue regeneration after myocardial infarction injury. RNA sequencing and chromatin methylation chromatin immunoprecipitation followed by sequencing were conducted to study the gene regulation in Abcg2-expressing ECs.RESULTS:In human and mouse vessels, ECs with higher ABCG2 expression (ABCECs) possess higher clonal proliferative potential and in vivo vessel-forming potential compared with mature ECs. These cells could clonally contribute to vessel formation in primary and secondary recipients after transplantation. These features of ABCECs meet the criteria of CRECs. Results from lineage tracing experiments confirm that Abcg2-expressing CRECs (AbcCRECs) contribute to arteries, veins, and capillaries in cardiac tissue development and vascular tissue regeneration after myocardial infarction. Transcriptome and epigenetic analyses reveal that a gene expression signature involved in angiogenesis and vessel development is enriched in AbcCRECs. In addition, various angiogenic genes, such as Notch2 and Hey2, are bivalently modified by trimethylation at the 4th and 27th lysine residue of histone H3 (H3K4me3 and H3K27me3) in AbcCRECs.CONCLUSIONS:These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction.

Tissue nanotransfection (TNT) topically delivers Etv2, Foxc2, and Fli1 (EFF) plasmids increasing vasculogenic fibroblasts (VF) and promoting vascularization in ischemic murine skin. Human dermal fibroblasts respond to EFF nanoelectroporation with elevated expression of endothelial genes in vitro, which is linked to increased ten-eleven translocase 1/2/3 (TET) expression. Single cell RNA sequencing dependent validation of VF induction reveals a TET-dependent transcript signature. TNTEFF also induces TET expression in vivo, and fibroblast-specific EFF overexpression leads to VF-transition, with TET-activation correlating with higher 5-hydroxymethylcytosine (5-hmC) levels in VF. VF emergence requires TET-dependent demethylation of endothelial genes in vivo, enhancing VF abundance and restoring perfusion in diabetic ischemic limbs. TNTEFF improves perfusion and wound closure in diabetic mice, while increasing VF in cultured human skin explants. Suppressed in diabetes, TET1/2/3 play a critical role in TNT-mediated VF formation which supports de novo blood vessel development to rescue diabetic ischemic tissue. Tissue nanotransfection (TNT) achieves non-viral vasculogenic reprogramming of the skin. Here the authors show that upregulation of TET1/2/3, limited under diabetic conditions, is effective in markedly improving TNT-induced vasculogenic outcomes thus rescuing ischemic diabetic tissue at risk.

Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. The endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the artery-to-capillary-to-vein axis. Although our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site- specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease.

Our recent work reported on the identification of the vasculogenic fibroblast (VF) that is capable of generating new blood vessels during tissue repair (Pal et al. Nat Com, 2023). While VF are physiologic and injury-inducible, inducible VF generation is blunted under conditions of diabetes. We report that such barrier may be overcome by the induction of fibroblast Etv2, Fli1 and Foxc2 (EFF) using tissue nanotransfection (TNT) technology (PMIDs: 28785092, 34837085, 35819852). To understand the mechanisms underlying VF function and significance, we developed EFFfl/fl COL1A2creER mice. In these mice, EFF regions in fibroblasts (COL1A2+ cells) are induced in response to tamoxifen. To gain insight into the significance of VF in vivo, hind limb ischemia (HLI) studies were performed. Significant overexpression of EFF was noted in laser capture dissected dermal fibroblast tissue elements of tamoxifen treated HLI mice (n=6, P < 0.05). Tamoxifen treatment enhanced hindlimb perfusion, as determined by laser speckle perfusion imaging (Perimed Inc.), in HLI mice (n=7, P < 0.05). This rescue was associated with increased abundance of COL1A2+VWF+ VF as compared to control (EFFfl/fl) (n=7, P<0.05). These favorable rescue findings were reproduced in diabetic (db/db) mice subjected to HLI (n=7, P<0.05). In conclusion, vasculogenic reprogramming in vivo is capable rescuing ischemic tissue under both nondiabetic and diabetic conditions. Disclosure S.K. Mohanty: None. K. Singh: None. M. Kumar: None. S.S. Verma: None. S. Roy: None. C.K. Sen: Consultant; SouthWest Technologies. Board Member; VisopalExo. Consultant; Vomaris Inc. S.C. Gnyawali: None. M.C. Yoder: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK136814, DK128845, DK135447, DK125835); Department of Defense (W81XWH-21-1-0033, W81XWH-22-1-0146)

Background & Aim Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity and is characterized by impaired lung development. Approximately 25-30% of infants diagnosed with BPD are estimated to develop pulmonary hypertension (PH) which doubles the risk of mortality. BPD-PH is characterized by abnormal vascular remodeling, alveolar diffusion impairment, and rarefication of pulmonary vessels resulting in increased pulmonary vascular resistance and right ventricular hypertrophy (RVH). Umbilical cord blood (UCB)-endothelial colony forming cells (ECFCs), a subset of endothelial progenitor cells with self-renewal and de novo vasculogenic capacity, enhance lung vascular growth and prevent hyperoxic neonatal lung injury in experimental BPD. Human induced pluripotent stem cell (hiPSC)-derived vasculogenic progenitor cells (hiPSC-VPCs) share similar characteristics with UCB-ECFCs and provide a nearly limitless source of allogeneic cells. We hypothesized that hiPSC-VPCs promote lung vascular growth and attenuate BPD-PH. Methods, Results & Conclusion In this study, we explored the use of hiPSC-VPCs in a neonatal hyperoxic mouse BPD model. Immuno-compromised Rag-/- mice pups, exposed to 85% oxygen from post-natal (P) day 4 to 14, received a single IV injection of 18E6/kg of iPSC-VPCs at P14 followed by a return to room air. Additionally, a dose-escalation study was conducted with IV injection of three different doses of iPSC-VPCs (10E6/kg, 20E6/kg, 40E6/kg) at P14. PH onset, lung function, and morphology were assessed at P27/P28 in both studies. Compared to untreated mice, the 18E6/kg dose of hiPSC-VPCs demonstrated significant improvements in pulmonary acceleration time/pulmonary ejection time (0.23±0.02 versus 0.26±0.03, P<0.01) and attenuated RVH (0.44±0.13 versus 0.28±0.07, P<0.0001). No significant differences were observed in lung function (0.017±0.01 versus 0.018±0.01, P>0.1) and lung morphology (53.2±4.1 versus 50±2.6, P>0.1). In the dose-escalation study, all three doses of hiPSC-VPCs attenuated RVH compared to untreated mice (0.36±0.07 versus 0.30±0.03, P>0.05, 0.26±0.04, P<0.0001, 0.28±0.05, P<0.05). No significant differences were observed in lung function (0.019±0.01 versus 0.017±0.01, P>0.1, 0.017± 0.01, P>0.1, 0.017±0.01, P>0.1) and lung morphology (58.45±5.1 versus 55.39± 7.7, P>0.1, 51.28±3.7, P>0.1, 57.36±5.9, P>0.1). In summary, hiPSC-VPCs therapy may offer a regenerative approach for neonatal PH.

Most circulating endothelial cells are apoptotic, but rare circulating endothelial colony-forming cells (C-ECFCs), also known as blood outgrowth endothelial cells, with proliferative and vasculogenic activity can be cultured; however, the origin and naive function of these C-ECFCs remains obscure. Herein, detailed lineage tracing revealed murine C-ECFCs emerged in the early postnatal period, displayed high vasculogenic potential with enriched frequency of clonal proliferative cells compared with tissue-resident ECFCs, and were not committed to or derived from the BM hematopoietic system but from tissue-resident ECFCs. In humans, C-ECFCs were present in the CD34bright cord blood mononuclear subset, possessed proliferative potential and in vivo vasculogenic function in a naive or cultured state, and displayed a single cell transcriptome sharing some umbilical venous endothelial cell features, such as a higher protein C receptor and extracellular matrix gene expression. This study provides an advance for the field by identifying the origin, naive function, and antigens to prospectively isolate C-ECFCs for translational studies.

Human-induced pluripotent stem cells (hiPSCs) cells have the proliferative potential and ability to differentiate into numerous cell types [...]

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.

Objectives: Critical Limb Threatening Ischemia (CLTI) is a health care priority with a 50% increase in amputations from 2010 to 2018. Unlike other cell products that simply secrete pro-angiogenic growth factors Induced pluripotent stem cells (iPSC) derived mesodermal cells can differentiate into endothelial and smooth muscle cells and form functioning blood vessels in vivo . We discuss the effects of intramuscular injection of human iPSC mesodermal cells (VSC100) on limb perfusion and limb necrosis. Methods: IPSCs were produced using methods including Yamanaka factors (Broxmeyer H. et al [21393480}). iPSC cells were differentiated using sequential Activin A, BMP4, VEGF, and FGF2 to promote mesodermal and endothelial cell (EC) differentiation. Murine CLTI models were created via excision of the common femoral artery in male BALB-C Nude mice, aged 6-8 weeks. After creation of the CLTI model mice were randomized to 1x10 6 VSC100 cells labeled with td-tomato (red fluorescence) or vehicle control injected into the gastrocnemius and gracilis muscles (N=10/group) at 7-14 days postinduction of ischemia. Limb perfusion was measured with Laser Speckle Contrast Imaging (LSCI) up to Day 64, qualitative analysis of VSC100 differentiation into capillaries was assessed with immunohistochemical staining for Isolectin B4 and human CD31 and confocal microscopy to detect td-tomato. Degree of tissue loss of the index limb was quantified with established necrosis scores. Results: VSC100 treated mice showed a significant increase in limb perfusion compared to controls, average percent blood flow/contralateral limb ratios of 45% in treated groups compared to 25% in control (p < 0.001) at day 64 (Figure 1). Limb necrosis scores showed necrosis was statistically significantly reduced in iPSC treated versus vehicle control treated groups (Figure 2). Immunohistochemical analysis showed qualitative significant deposition of cells of interest in our CLTI models. Conclusions: iPSC derived VSC100 is a novel approach to treating CTLI as the cells can form functioning blood vessels in ischemic skeletal muscle, improve tissue perfusion, and mitigate tissue necrosis. These early results are being used for FDA applications for clinical trials utilizing iPSCs in CLTI patients.

Hal E. Broxmeyer died December 8, 2021 at the age of 77. He was an outstanding experimental hematologist who likely will be most remembered scientifically as a pioneer in identifying human umbilical cord blood (UCB) as a source of transplantable hematopoietic stem and progenitor cells (HSPCs). He further translated this knowledge to cryopreserve and provide the first UCB unit for transplantation and successful engraftment in a patient with Fanconi anemia in Paris in 1988 under the guidance of Dr. Eliane Gluckman. The field of UCB transplantation expanded worldwide upon publication of the outcome of this first transplantation and an estimated 40,000 patients have subsequently received UCB transplants worldwide for hematologic disorders and malignancies. Such an impactful contribution early in his faculty career (8th year) may have been met with considerable distractions in deciding where to focus his laboratories’ future research efforts next. But not for Hal. He understood that to fully achieve the benefits of UCB for transplantation, scientists would need to deeply interrogate and understand the developmental and comparative biological properties of the UCB HSPCs in comparison with adult bone marrow HSPCs. He pursued such investigations in a strategic, passionate, and tenacious fashion. Over his career, Hal not only became a leader in UCB biology but was also valued as an advisor for stem cell research institutes, cancer centers, and biotechnology companies for his expert knowledge in the broader areas of leukemic stem cell biology and pluripotent stem cells. Hal was born in Brooklyn, New York, on November 27, 1944. He is known for his loyalty to the Brooklyn Dodgers and it is said that he never cheered for another baseball team when the Dodgers left for Los Angeles in 1957. Hal enrolled in Brooklyn College as a math major but became intrigued with science and upon graduation obtained an MS degree from Long Island University Brooklyn Campus. He was awarded his PhD at New York University in 1973 and then completed a post-doctoral fellowship at Queens University, Kingston, Ontario before accepting an Assistant Professor position at Sloan Kettering Institute in New York. He began his research career focused on identifying negative regulators of hematopoiesis. However, in 1982, Dr. Edward Boyse called Hal into his office to discuss the idea of potentially using UCB in some clinical fashion; perhaps collecting the mature granulocytes, red blood cells, or platelets as a transfusion product. Hal was aware of published papers suggested UCB may contain hematopoietic progenitor cells and perhaps even stem cells, but whether UCB could possess potential for clinical stem cell transplantation remained a curiosity. Thus began a collaboration that would grow to include Drs. Judith Bard, Lewis Thomas, Harvey Cantor, Gordon Douglas, Arleen Auerbach, and Elian Gluckman (among others) and that would coalesce around the idea of using UCB for transplantation. Details of this story are intriguing for the interested reader (Ballen et al., 2013Ballen K.K. Gluckman E. Broxmeyer H.E. Umbilical cord blood transplantation: the first 25 years and beyond.Blood. 2013; 122: 491-498Crossref PubMed Scopus (472) Google Scholar). In 1983, Hal was recruited to Indiana University School of Medicine in Indianapolis, Indiana, where he pursued critical feasibility studies to demonstrate UCB possessed sufficient HSPCs for clinical transplantation. These experiments launched an astonishing career of investigation into the role of growth factors, chemokines, and cytokines on the proliferation, differentiation, migration, survival, homing, and mobilization of HSPCs that would culminate in >830 peer-reviewed publications, hundreds of book chapters, and numerous edited books and editorials. Such productivity would make one think Hal was fiercely independent and laser-focused with no time for administration, mentoring, or teaching. But, in fact, he was a model faculty member and collaborator with an intense interest in promoting the development of students, post-doctoral fellows, and young faculty members. Hal served as the first director of the Walther Oncology Center and chair of Microbiology and Immunology for many years. At the time of his death, he was an IU Distinguished Professor, Mary Margaret Walther Professor Emeritus, and professor of Microbiology and Immunology at IU School of Medicine and senior advisor to the director of the Indiana University Melvin and Bren Simon Comprehensive Cancer Center. For many years, Hal was principal investigator on multiple training grants in hematopoiesis and cell and gene therapy for PhD students and post-doctoral fellows. He was also the lead principal investigator on several hematopoiesis program project and cancer grants. He was an inspiring teacher who won many teaching awards from the PhD students. He served his research colleagues as an editor or on the editorial board of numerous journals and on many National Institutes of Health study sections as a member or chair, and he led several societies as president. These societies include the American Society of Hematology and the International Society for Experimental Hematology. In sum, “Hal was a force of nature,” recalls his colleague and friend, Dr. Patrick (Pat) Loehrer. In Hal’s own words, he would tell you he loved research and that is why even while serving on all the School of Medicine and national and international society administrative positions he continued to work in his laboratory daily. His favorite and somewhat exclusive task was to score the colony counts in the hematopoietic progenitor cell colony forming assays for the lab. It was not uncommon to have an appointment with Hal and, upon entering his office, find him surrounded by columns of Lux culture dishes stacked on tissue culture incubator shelves. He would be peering through his 40-year-old Olympus microscope counting the numbers and types of colonies formed. He always wanted to see the results of the studies before anyone else in the lab. He would comment that he enjoyed seeing how the results addressed or refuted the hypotheses posed and this would stimulate him to ask the next set of questions and set up more experiments. Hal was first diagnosed with thyroid cancer in 2013. He kept his illness from nearly everyone but his family and his lab members. He recuperated quickly and was soon back to his high level of activity. Then he had a recurrence that affected his vocal cords and required tracheostomy, radiation therapy, and other treatments. The combination of treatments made him very tired. He did not waste time decrying his ailment or retreating into privacy but continued to give lectures (with much effort). He noted to his colleague Pat several weeks before his death, “Today, I feel better than I have for weeks. I hope it lasts, but I am thankful for each good day that I have.” As another colleague, Dr. Christi Orschell, commented upon considering Hal’s legacy, “So much good about that man.” The author wishes to thank Dr. Patrick Loehrer and Dr. Christi Orschell from Indiana University School of Medicine for their contributions to this tribute.

Given the strong association between impaired renal microcirculation and disease states, significant attention has been directed toward understanding renal vascular repair. Endothelial progenitor cells (EPCs) have been studied in different cardiovascular models, including preclinical models of kidney disease. However, as this field of study has evolved over the last 20 years, it has been hampered by inconsistent definitions surrounding the term EPC. This chapter attempts to provide relevant background regarding vascular injury and impaired regenerative responses in the setting of either acute kidney injury (AKI) or chronic kidney disease (CKD). We provide an updated discussion on different types of cells previously classified broadly as EPCs and discuss potential interactions of these cells in vascular repair. Using this framework, we present a hypothesis by which the activity of intrinsic vascular progenitors represents a central element of disease progression. Finally, we summarize evidence that cells with vasculoreparative properties may represent potential therapies in disease states.

HomeCirculation ResearchVol. 130, No. 6Readily Available Tissue-Engineered Vascular Grafts Derived From Human Induced Pluripotent Stem Cells Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBReadily Available Tissue-Engineered Vascular Grafts Derived From Human Induced Pluripotent Stem Cells Jiesi Luo, Lingfeng Qin, Jinkyu Park, Mehmet H. Kural, Yan Huang, Xiangyu Shi, Muhammad Riaz, Juan Wang, Matthew W. Ellis, Christopher W. Anderson, Yifan Yuan, Yongming Ren, Mervin C. Yoder, George Tellides, Laura E. Niklason and Yibing Qyang Jiesi LuoJiesi Luo https://orcid.org/0000-0002-3041-7283 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) , Lingfeng QinLingfeng Qin Surgery (L.Q., G.T.) , Jinkyu ParkJinkyu Park Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) , Mehmet H. KuralMehmet H. Kural Anesthesiology (M.H.K., J.W., Y.Y., L.E.N.) Vascular Biology and Therapeutics Program, Yale School of Medicine (M.H.K., J.W., Y.Y., G.T., L.E.N., Y.Q.). , Yan HuangYan Huang Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) , Xiangyu ShiXiangyu Shi Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) Cardiovascular Medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (X.S.). , Muhammad RiazMuhammad Riaz https://orcid.org/0000-0002-0311-8392 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) , Juan WangJuan Wang https://orcid.org/0000-0003-0219-4200 Anesthesiology (M.H.K., J.W., Y.Y., L.E.N.) Vascular Biology and Therapeutics Program, Yale School of Medicine (M.H.K., J.W., Y.Y., G.T., L.E.N., Y.Q.). , Matthew W. EllisMatthew W. Ellis Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) Cellular and Molecular Physiology (M.W.E.) , Christopher W. AndersonChristopher W. Anderson Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) Pathology, Yale School of Medicine (C.W.A., Y.Q.). , Yifan YuanYifan Yuan Anesthesiology (M.H.K., J.W., Y.Y., L.E.N.) Vascular Biology and Therapeutics Program, Yale School of Medicine (M.H.K., J.W., Y.Y., G.T., L.E.N., Y.Q.). , Yongming RenYongming Ren Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) , Mervin C. YoderMervin C. Yoder https://orcid.org/0000-0003-4354-7631 Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, U.S.A. (M.C.Y.). , George TellidesGeorge Tellides Surgery (L.Q., G.T.) Vascular Biology and Therapeutics Program, Yale School of Medicine (M.H.K., J.W., Y.Y., G.T., L.E.N., Y.Q.). , Laura E. NiklasonLaura E. Niklason Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) Anesthesiology (M.H.K., J.W., Y.Y., L.E.N.) Biomedical Engineering (L.E.N.) Vascular Biology and Therapeutics Program, Yale School of Medicine (M.H.K., J.W., Y.Y., G.T., L.E.N., Y.Q.). and Yibing QyangYibing Qyang Correspondence to: Yibing Qyang, PhD, Internal Medicine, Yale University School of Medicine, Room 773A, 300 George Street, New Haven, CT 06511. Email E-mail Address: [email protected] https://orcid.org/0000-0002-0332-2449 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, U.S.A. (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., Y.Q.) Yale Stem Cell Center (J.L., J.P., Y.H., X.S., M.R., M.W.E., C.W.A., Y.R., L.E.N., Y.Q.) Vascular Biology and Therapeutics Program, Yale School of Medicine (M.H.K., J.W., Y.Y., G.T., L.E.N., Y.Q.). Pathology, Yale School of Medicine (C.W.A., Y.Q.). Originally published22 Feb 2022https://doi.org/10.1161/CIRCRESAHA.121.320315Circulation Research. 2022;130:925–927is related toMeet the First AuthorsOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: February 22, 2022: Ahead of Print Meet the First Author, see p 812Readily available tissue-engineered vascular grafts (TEVGs) have been developed as a promising, off-the-shelf therapeutic option for cardiovascular interventions. By culturing human donor-derived primary vascular smooth muscle cells on biodegradable scaffolds coupled with tissue decellularization, mechanically strong, decellularized TEVGs can be developed as a readily available therapy for clinical use.1 However, application of primary cells may hinder current TEVGs in treating obstructive atherosclerosis, which occurs most commonly in small-caliber (luminal diameter, 2–4 mm) vessels (eg, coronary arteries) and leads to devastating symptomatic end-organ ischemia. Donor-derived primary cells may display limited in vitro expandability and variation in cellular function, leading to batch-to-batch variation in TEVG quality.1 Further, small-caliber TEVGs require luminal coating by autologous primary endothelial cells (ECs) to prevent blood clotting and thrombogenesis, and the long patient wait-time (≈23 days) required make these TEVGs less practical.1 To address these challenges, immunologically modified human induced pluripotent stem cells (hiPSCs) could serve as an alternative source for deriving vascular cells in large quantities with comparable quality for TEVG generation.2 We have previously reported cell-based TEVGs developed using hiPSCs (hiPSC-TEVGs; Figure [A], i).2 However, generation of small-caliber (luminal diameter, 2–4 mm), decellularized hiPSC-TEVGs that support luminal endothelialization as a readily available therapy for treating obstructive vascular diseases remains unexplored.Download figureDownload PowerPointFigure. Characterizing readily available human induced pluripotent stem cell (hiPSC)-tissue-engineered vascular grafts (TEVGs).A, Schematic for research (i) and representative, decellularized hiPSC-TEVG images (ii). B, Characterizing hiPSC-TEVGs in vitro: assessing DNA (i), cellular proteins (α-SMA [α-smooth muscle actin], β-actin, and GAPDH; ii), collagen (iii), histological and VSMC (α-SMA, CNN1 [calponin 1], and MYH11 [myosin heavy chain 11]) markers (iv), and mechanical properties (v; rat aorta as reference). C, Evaluating decellularized hiPSC-TEVGs in nude rats for 30 days: a representative image of explanted grafts (i), ultrasonographic analysis (ii), and histological (human-specific surface antigen [HLA-A]) analyses of explanted grafts (iii). D, Assessing decellularized hiPSC-TEVGs that support luminal endothelialization in flow bioreactors (5 dynes/cm2): graft endothelialization schematic (i), endothelial marker immunostaining (CD31, eNOS; ii), luminal endothelial cell (EC) coverage rates (iii), and luminal surface via scanning electron microscopy (iv). E, Characterizing hiPSC-EC-based endothelialization of decellularized rat allograft aortas (58.4 dynes/cm2): immunostaining of endothelial and basement membrane (COLIV [collagen type IV] and laminin) markers before implantation (i), H&E staining (ii), and immunostaining of CD31/HLA in day-7 and -30 explanted grafts (iii). Images from 3 biological replicates, with typical morphological and histological presentation, were selected as representative images. Immunostaining was performed vs IgG controls. Asterisks (B, iv; C, iii; D, ii, and E), graft lumen; pound (E, ii), thrombosis; dashed lines, graft boundary (B, iv) or sectioning position (C, i); arrow heads, discontinued COLIV and laminin staining (E, i) or ECs on graft lumen (E, ii). Data were analyzed via Mann-Whitney U test (B, i; B, iii; and B, v) or Kruskal-Wallis test (C, ii); n indicates numbers of biological replicates; and NS, not significant. *P<0.05.We first decellularized hiPSC-TEVGs (Figure [A], i) using our previously reported approach.3 Cell-based hiPSC-TEVGs (luminal diameter, 3.2 mm) were generated and decellularized (Figure [A], ii). DNA and cellular proteins were effectively removed from hiPSC-TEVGs (Figure [B], i and [B], ii), while the collagen content was preserved postdecellularization (Figure [B], iii). Histological analysis confirmed the removal of cells, retention of collagen, and absence of calcification in decellularized hiPSC-TEVGs (Figure [B], iv). Mature elastin fibers were not observed (data not shown), as is characteristic of current TEVG generation.2 Moreover, decellularized hiPSC-TEVGs displayed robust mechanical properties (rupture pressure: 1203.5±116.5 mm Hg; suture retention strength: 123.8±12.1 g; n=4; Figure [B], v), which were not statistically different from those of cell-based hiPSC-TEVGs (rupture pressure: 1419.0±174.4 mm Hg [P=0.20]; suture retention strength: 157.5±16.5 g [P=0.20]; n=4).2 These results suggest hiPSC-TEVGs are efficiently decellularized without significantly reducing mechanical strength.We subsequently implanted decellularized hiPSC-TEVGs into nude rats as interposition aortic grafts for 30 days.2 Grafts remained patent without rupture when explanted and did not present aneurysmal dilation or abnormal elongation during implantation (Figure [C], i and [C], ii). Luminal diameters of implanted grafts slightly increased after implantation but appeared stabilized after week 3, potentially due to effective recellularization of implanted grafts. Limited thrombogenesis was observed in explanted grafts, which might be attributed to secondary flow and flow reversal due to an imperfect size match between grafts and host vessels in this proof-of-principle study. Histological analysis revealed implanted grafts were repopulated with host cells and maintained collagen deposition without apparent calcification (Figure [C], iii). A substantial number of cells repopulating the graft wall expressed vascular smooth muscle cell-specific proteins but were negative for human-specific surface antigens, confirming their vascular smooth muscle cell identities and host origin (Figure [C], iii). These data suggest decellularized hiPSC-TEVGs were implantable as aortic grafts and supported efficient recellularization.We next explored endothelializing decellularized hiPSC-TEVGs using hiPSC-derived ECs (hiPSC-ECs)4 under shear stress. Endothelialized grafts displayed efficient luminal surface coverage with hiPSC-ECs aligned to the direction of flow (Figure [D], i through [D], iv). We then implanted endothelialized grafts into nude rats as aortic grafts for 30 days. Explanted grafts remained patent, though with appreciable thrombogenesis (data not shown), likely due to disturbed blood flow caused by the larger size of hiPSC-TEVGs compared with the rat abdominal aorta. To evaluate endothelialization in more similarly size-matched grafts, hiPSC-EC-coated and uncoated decellularized rat allograft aortas were implanted as aortic grafts into nude rats (Figure [E], i). While endothelialized allografts were patent, with no appreciable thrombogenesis 7 and 30 days postimplantation, nonendothelialized grafts displayed evident thrombogenesis (Figure [E], ii). Moreover, endothelialized grafts showed an endothelial population containing both hiPSC-ECs and rat host ECs 7 days postimplantation that shifted entirely to rat ECs by 30 days postimplantation (Figure [E], iii). In contrast, ECs were barely detectable in nonendothelialized grafts (Figure [E], iii). These results suggest hiPSC-ECs may prevent graft thrombogenesis via providing immediate EC function and expediting recruitment of and endothelialization by host ECs potentially through the deposition of basement membrane proteins collagen IV and laminin (Figure [E], i). Future efforts are warranted to understand the gradual replacement of hiPSC-ECs in grafts by host rat ECs, the communication between these two types of ECs, and the source of host ECs that endothelialize the grafts (eg, from local vessels, circulating ECs, or circulating endothelial progenitor cells).5 Future work is also required to apply large animal models that mimic the hemodynamics and dimensions of human vessels to evaluate the long-term antithrombotic and antistenotic effects of current endothelialized grafts. Additionally, universal hiPSC-ECs will be used to develop TEVGs immunocompatible to many patients.2 In summary, this study represents the first readily available, small-caliber hiPSC-TEVGs, setting the stage for using novel, allogeneic vascular grafts as an effective future therapy for patients with cardiovascular diseases.Article InformationAcknowledgmentsRaw research data and full methods will be made available on request from the corresponding author.Sources of FundingThis study was supported by DOD W81XWH1910557, NIH R01HL116705, NIH R01HL150352, and NIH R01HL155411 (all to Y.Q.), The American Heart Association Postdoctoral Fellowship Awards 19POST34450100 (to J.L.), 19POST34381048 (to M.H.K.), and 20POST35210709 (to Y.Y.), Ruth L. Kirschstein Predoctoral Individual National Research Service Award NIH F31HL143928 (to C.W.A.), NIH F31HL143924, and NIH T32GM0007324 (to M.W.E.).DisclosuresNone.Nonstandard Abbreviations and AcronymsECendothelial cellhiPSChuman induced pluripotent stem cellhiPSC-EChiPSC-derived EChiPSC-TEVGTEVGs developed using hiPSCTEVGtissue-engineered vascular graftFootnotes*J. Luo, L. Qin, J. Park, and M.H. Kural contributed equally.For Sources of Funding and Disclosures, see page 926.Correspondence to: Yibing Qyang, PhD, Internal Medicine, Yale University School of Medicine, Room 773A, 300 George Street, New Haven, CT 06511. Email yibing.[email protected]eduReferences1. Dahl SL, Kypson AP, Lawson JH, Blum JL, Strader JT, Li Y, Manson RJ, Tente WE, DiBernardo L, Hensley MT, et al.. Readily available tissue-engineered vascular grafts.Sci Transl Med. 2011; 3:68ra9. doi: 10.1126/scitranslmed.3001426CrossrefMedlineGoogle Scholar2. Luo J, Qin L, Zhao L, Gui L, Ellis MW, Huang Y, Kural MH, Clark JA, Ono S, Wang J, et al.. Tissue-engineered vascular grafts with advanced mechanical strength from human iPSCs.Cell Stem Cell. 2020; 26:251–261.e8. doi: 10.1016/j.stem.2019.12.012CrossrefMedlineGoogle Scholar3. Park J, Anderson CW, Sewanan LR, Kural MH, Huang Y, Luo J, Gui L, Riaz M, Lopez CA, Ng R, et al.. Modular design of a tissue engineered pulsatile conduit using human induced pluripotent stem cell-derived cardiomyocytes.Acta Biomater. 2020; 102:220–230. doi: 10.1016/j.actbio.2019.10.019CrossrefMedlineGoogle Scholar4. Prasain N, Lee MR, Vemula S, Meador JL, Yoshimoto M, Ferkowicz MJ, Fett A, Gupta M, Rapp BM, Saadatzadeh MR, et al.. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells.Nat Biotechnol. 2014; 32:1151–1157. doi: 10.1038/nbt.3048CrossrefMedlineGoogle Scholar5. McDonald AI, Shirali AS, Aragón R, Ma F, Hernandez G, Vaughn DA, Mack JJ, Lim TY, Sunshine H, Zhao P, et al.. Endothelial regeneration of large vessels is a biphasic process driven by local cells with distinct proliferative capacities.Cell Stem Cell. 2018; 23:210–225.e6. doi: 10.1016/j.stem.2018.07.011CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMeet the First AuthorsCirculation Research. 2022;130:810-813 March 18, 2022Vol 130, Issue 6Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.121.320315PMID: 35189711 Originally publishedFebruary 22, 2022 Keywordstransplantationendothelial cellstissue engineeringinduced pluripotent stem cellsPDF download Advertisement SubjectsStem Cells

Stem Cells and DevelopmentVol. 31, No. 3-4 In MemoriamHal Broxmeyer, PhD (November 27, 1944–December 8, 2021)Eliane Gluckman, Christie M. Orschell, D. Wade Clapp, Edward Srour, Mervin C. Yoder, and John E. WagnerEliane GluckmanSearch for more papers by this author, Christie M. OrschellSearch for more papers by this author, D. Wade ClappSearch for more papers by this author, Edward SrourSearch for more papers by this author, Mervin C. YoderSearch for more papers by this author, and John E. WagnerSearch for more papers by this authorPublished Online:10 Feb 2022https://doi.org/10.1089/scd.2022.29010.memAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 31Issue 3-4Feb 2022 InformationCopyright 2022, Mary Ann Liebert, Inc., publishersTo cite this article:Eliane Gluckman, Christie M. Orschell, D. Wade Clapp, Edward Srour, Mervin C. Yoder, and John E. Wagner.Hal Broxmeyer, PhD (November 27, 1944–December 8, 2021).Stem Cells and Development.Feb 2022.35-37.http://doi.org/10.1089/scd.2022.29010.memPublished in Volume: 31 Issue 3-4: February 10, 2022Online Ahead of Print:January 24, 2022PDF download

BACKGROUND:Endothelial colony-forming cells (ECFCs) contribute to postnatal vasculogenesis. In venous thromboembolic disease (VTD), they are functionally abnormal and produce high concentrations of TNF-α. OBJECTIVE:To analyze the TNF-α signaling pathway and its relationship with the expression of cell-cycle regulators. METHODS:Mononuclear cells (MNCs) were collected from the peripheral blood of 20 healthy human volunteers (controls) and 30 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We analyzed the relative quantification of the gene transcripts of TNF, NFkB1, PLAU, HMOX1, GSS, eNOS, CDKN1A, and CDKN1B through quantitative RT-PCR (qRT-PCR assays). Identification of NF-κB and activated targets of each pathway: NF-κB (Ser536); IκBα (Ser32/Ser36); p38 (Thr180/Tyr182) JNK (Thr183/Tyr185), p53 and cell-cycle regulators: p16, p18, p21, p27, p57, Cyclin D, Cyclin E, Cyclin A, Cyclin B, CDK2, CDK4; cell-cycle status was determined by KI-67 and 7-AAD. Cells were analyzed with flow cytometry and the FlowJo vX software. RESULTS:In ECFCs from VTD patients, TNF-α receptor and NFkB were overexpressed and hyper-phosphorylated; eNOS and HMOX1 were down-regulated; cell-cycle regulators (p53, p18, p21) were elevated. In addition, the cell cycle was locked in the G2 phase. CONCLUSIONS:Our results strongly suggest that these molecular alterations in the pathway of TNF-α and cell cycle regulation induce endothelial dysfunction, reduced proliferation potential and vascular regeneration, and consequently, the occurrence of new thrombotic events.

Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR + CD56 + APLNR + (KNA + ) expression. KNA + cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA + cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice ( db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA + cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA + cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor–derived KNA + cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide “proof of principle” that KNA + cells restore perfusion and correct vascular dysfunction in db/db mice.