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Human induced pluripotent stem cells (hiPSCs) not only provide an abundant source of vascular cells for potential therapeutic applications in vascular disease but also constitute an excellent model for understanding the mechanisms that regulate the differentiation and the functionality of vascular cells. Here, we reported that myocyte enhancer factor 2C (MEF2C) transcription factor, but not any other members of the MEF2 family, was robustly upregulated during the differentiation of vascular progenitors and endothelial cells (ECs) from hiPSCs. Vascular endothelial growth factors (VEGF) strongly induced MEF2C expression in endothelial lineage cells. The specific upregulation of MEF2C during the commitment of endothelial lineage was dependent on the extracellular signal regulated kinase (ERK). Moreover, knockdown of MEF2C with shRNA in hiPSCs did not affect the differentiation of ECs from these hiPSCs, but greatly reduced the migration and tube formation capacity of the hiPSC-derived ECs. Through a chromatin immunoprecipitation-sequencing, genome-wide RNA-sequencing, quantitative RT-PCR, and immunostaining analyses of the hiPSC-derived endothelial lineage cells with MEF2C inhibition or knockdown compared to control hiPSC-derived ECs, we identified TNF-related apoptosis inducing ligand (TRAIL) and transmembrane protein 100 (TMEM100) as novel targets of MEF2C. This study demonstrates an important role for MEF2C in regulating human EC functions and highlights MEF2C and its downstream effectors as potential targets to treat vascular malfunction-associated diseases.

Diabetic ischemic wound treatment remains a critical clinical challenge. Neovascularization plays a significant role in wound healing during all stages of the tissue repair process. Strategies that enhance angiogenesis and neovascularization and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic conditions. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates vascular endothelial growth factor (VEGF) receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a multi-functional pro-angiogenic molecule by grafting LXW7 and collagen-binding peptides (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY, and further employed this multi-functional molecule to functionalize collagen-based extracellular matrix (ECM) scaffolds. We confirmed that LXW7-DS-SILY modification significantly promoted EPC attachment and growth on the ECM scaffolds in vitro and supported EPC survival in vivo in the ischemic environment. When applied in an established Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model, LXW7-DS-SILY-functionalized ECM scaffolds loaded with EPCs significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients., Graphical abstract Schematics for the fabrication of ligand modified scaffolds.Image 1, Highlights • Diabetic foot ulcers represent a significant healthcare burden. • Neovascularization plays a significant role in all stages of the wound healing process. • Endothelial progenitor cells (EPCs) have the potential to facilitate neovascularization. • LXW7-DS-SILY promoted EPC attachment and growth on ECM scaffolds in vitro. • LXW7-DS-SILY improved neovascularization and wound healing in ZDF rat ischemic model.

Nanoparticle-based drug delivery systems have the potential to revolutionize medicine but their low vascular permeability and rapid clearance by phagocytic cells have limited their medical impact. Nanoparticles delivered at thein uterostage have the potential to overcome these key limitations, due to the high rate of angiogenesis and cell division in fetal tissue, and the under-developed immune system. However, very little is known about nanoparticle drug delivery at the fetal stage of development. In this report, using Ai9 CRE reporter mice, we demonstrate that lipid nanoparticle (LNP) mRNA complexes can deliver mRNA for gene editing enzymesin uteroafter an intrahepatic injection, and can access and edit major organs, such as the heart, the liver, kidneys, lungs and the gastrointestinal tract with remarkable efficiency and low toxicity. In addition, we show here that Cas9 mRNA and sgRNA complexed to LNPs were able to edit the fetal organsin uteroafter an intrahepatic injection. These experiments demonstrate the possibility of non-viral delivery of gene editing enzymesin uteroand nanoparticle drug delivery has great potential for delivering macromolecules to organs outside of the liverin utero, which provides a promising strategy for treating a wide variety of devastating genetic diseases before birth.

Hemophilia A (HA) is a bleeding disorder characterized by spontaneous and prolonged hemorrhage. The disease is caused by mutations in the coagulation factor 8 gene (F8) leading to factor VIII (FVIII) deficiency. Since FVIII is primarily produced in endothelial cells (ECs) in a non‐diseased human being, ECs hold great potential for development as a cell therapy for HA. We showed that HA patient‐specific induced pluripotent stem cells (HA‐iPSCs) could provide a renewable supply of ECs. The HA‐iPSC‐derived ECs were transduced with lentiviral vectors to stably express the functional B domain deleted F8 gene, the luciferase gene, and the enhanced green fluorescent protein gene (GFP). When transplanted intramuscularly into neonatal and adult immune deficient mice, the HA‐iPSC‐derived ECs were retained in the animals for at least 10‐16 weeks and maintained their expression of FVIII, GFP, and the endothelial marker CD31, as demonstrated by bioluminescence imaging and immunostaining, respectively. When transplanted into HA mice, these transduced HA‐iPSC‐derived ECs significantly reduced blood loss in a tail‐clip bleeding test and produced therapeutic plasma levels (11.2%‐369.2%) of FVIII. Thus, our studies provide proof‐of‐concept that HA‐iPSC‐derived ECs can serve as a factory to deliver FVIII for the treatment of HA not only in adults but also in newborns., Schematic diagram of the experimental approach.

This study investigated the feasibility and efficiency of neuron-targeting hybrid placental mesenchymal stromal cell-derived extracellular vesicles (PMSC-EVs), engineered by membrane fusion with Targeted Axonal Import (TAxI) peptide modified, TrkB agonist 7,8-DHF-loaded liposomes for treatment of myelomeningocele (MMC) via intra-amniotic cavity administration. The prepared TAxI modified liposomes with 7,8-DHF were used to fuse with PMSC-EVs. Different fusion approaches were investigated and freeze-thaw-extrude method was found to be the optimal. The engineered PMSC-EVs had a uniform particle size and efficiently loaded 7,8-DHF. It also had typical markers of native EVs. Freeze-thaw-extrude process did not change the release profile of 7,8-DHF from engineered EVs compared to TAxI modified, 7,8-DHF loaded liposomes. The engineered EVs could elicit TrkB phosphorylation depending on the incorporation of 7,8-DHF while native EVs did not. The engineered EVs increased neurite outgrowth of apoptotic cortical neurons induced by staurosporine, suggesting that they exhibited neuroprotective function. In a rodent model of MMC, neuron-targeting, engineered EVs became an active targeting delivery system to MMC defect sites. Pups treated with engineered EVs had the lowest density of apoptotic cells and displayed a therapeutic outcome. The study suggests the potential use of engineered hybrid, active neuron-targeting EVs for the in utero treatment of MMC.

Xinxin Ren,1 Xiang Wang,2 Bi Peng,3 Qiuju Liang,2 Yuan Cai,3 Kewa Gao,3 Yongbin Hu,3 Zhijie Xu,3,4 Yuanliang Yan2 1Center for Molecular Medicine, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 2Department of Pharmacy, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 3Department of Pathology, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 4National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, People’s Republic of ChinaCorrespondence: Yuanliang YanDepartment of Pharmacy, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, People’s Republic of ChinaEmail yanyuanliang@csu.edu.cnPurpose: To explore the molecular profiles of transcriptional enhanced associate domain (TEAD) family in ovarian serous carcinoma (OSC).Methods: In this study, we use bioinformatics methods including GEPIA, GE-mini, Oncomine 3.0, Kaplan–Meier plotter, cBioPortal, WebGestalt, TIMER2.0 and DiseaseMeth2.0, and in vitro experimental RT-PCR to assess the expression profiles and prognostic significance of TEAD family in OSC.Results: According to the bioinformatics analysis, TEAD family was abnormally expressed in OSC. In terms of prognosis, Kaplan–Meier plotter indicated that OSC patients with high level of TEAD4 showed poor overall survival (OS), progression-free survival (PFS) and post progression survival (PPS). TEAD family also had significantly diagnostic values for OSC patients. Tumor Immune Estimation Resource (TIMER) algorithm indicated that TEAD family was significantly associated with different types of infiltrating immune cells, including B cells, macrophages, dendritic cells, neutrophils, CD8+ T cells and CD4+ T cells. Gene set enrichment analysis of TEAD family-associated coexpression genes was further explored. In in vitro experiments, the RT-PCR results showed the upregulated TEAD2/4 in OSC tissues and cells (A2780 and TOV112D). Moreover, decreased expression of TEAD2 could induce the ferroptosis through increasing the ROS accumulation.Conclusion: Thus, TEAD family correlated with the diagnosis, prognosis and immune infiltration in OSC. These results could provide comprehensive understanding of TEAD family in the diagnosis and prognosis of OSC patients.Keywords: TEAD family, Hippo pathway, ovarian serous carcinoma, expression profiles, prognosis, immune infiltration

Background: Alterations of mitogen-activated protein kinase kinase 1 (MEK1) are commonly associated with tumorigenesis, and MEK1 is thought to be a suitable targeted therapy for various cancers. However, abnormal MEK1 alterations and their relevant clinical implications are unknown. Our research comprehensively analyzed the MEK1 alteration spectrum and provided novel insight for targeted therapies. Methods: There were 7694 samples covering 32 types of cancer from The Cancer Genome Atlas (TCGA) database. They were used to conduct an integrative analysis of MEK1 expression, alterations, functional impacts and clinical significance. Results: There was a dramatic difference in the alteration frequency and distribution and clinical implications in 32 types of cancer from the TCGA. Skin cutaneous melanoma (SKCM) has the most alterations and has therapeutic targets located in the protein kinase domain, and the growing expression of SKCM is positively related to patient prognosis. MEK1 expression in lung adenocarcinoma (LUAD), kidney renal papillary cell carcinoma (KIRP), esophageal carcinoma (ESCA) and liver hepatocellular carcinoma (LIHC) is decreased, which is associated with better prognosis, while MEK1 expression in thymoma (THYM), stomach adenocarcinoma (STAD), kidney renal clear cell carcinoma (KIRC), testicular germ cell tumors (TGCTs) and head and neck squamous cell carcinoma (HNSC) is increased, which is associated with better prognosis. Mesothelioma (MESO) has the second highest alterations but has no therapy targets. Conclusion: This study provided a great and detailed interpretation of MEK1 expression, alterations and clinical implications in 32 types of cancer and reminded us to fill the gap in MEK1 research from a new perspective. Keywords: MEK1, gene mutation, gene alteration, outcome, pan-cancer

In early pregnancy, fetal skin wounds can heal quickly and undergo a transition period from scarless healing to scar formation. The aim of the present study was to identify potential biomarkers associated with scarless repair of cleft lips, in order to determine the intrinsic factors leading to scar formation in embryonic tissue. A stable model of cleft lip was established using microsurgery by constructing a wedge‑shaped cleft lip‑like defect in fetal rats at gestational age (GA) 16.5 and GA18.5. The GA16.5 and GA18.5groups were used to model scarless healing and scar formation, respectively. The fetuses were returned to the uterus following surgery, then removed 72h after the procedure. Macroscopic observation of the cleft defect and histological examination were carried out. Reverse transcription‑quantitative(RT‑q) PCR and parallel reaction monitoring(PRM) were used to detect mRNA and protein expression levels, respectively. The upper‑left lip completely healed 72h after surgery in the GA16.5group of fetal rats. However, this was not the case in the GA18.5group. Histological examination indicated new follicles visible under the epidermis of the scarlessgroup(GA16.5). Scarring was visible on the upper‑left cleft lip wound of the fetal rats in the GA18.5group. The expression of some growth and pro‑inflammatory factors, including TNF‑α, were also different between two groups. Label‑free quantification was used to identified differentially expressed proteins and five differentially expressed proteins (Smad4, Fabp5, S100a4, S100a8 and S100a9) were identified. The relative expression of these molecules at the mRNA and protein levels were measured using RT‑qPCR and PRM. These molecules may represent potential biomarkers for the scarless repair of fetal rat cleft lip wounds.

Diabetic ischemic wound treatment remains a critical clinical challenge. Strategies that enhance angiogenesis and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic condition. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates VEGF receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a pro-angiogenic molecule LXW7-DS-SILY by conjugating LXW7 to a collagen-binding proteoglycan mimetic DS-SILY and further employed this novel bifunctional ligand to functionalize extracellular matrix (ECM) scaffolds, promote neovascularization and accelerate ischemic wound healing. We established a Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model and found the wounds treated by LXW7-DS-SILY-functionalized ECM scaffolds, with or without EPCs, significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis. These functionalized ECM scaffolds also significantly promoted EPC attachment, growth and survival in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

BACKGROUND: Endothelial colony-forming cells (ECFCs) have been implicated in the process of vascularization, which includes vasculogenesis and angiogenesis. Vasculogenesis is a de novo formation of blood vessels, and is an essential physiological process that occurs during embryonic development and tissue regeneration. Angiogenesis is the growth of new capillaries from pre-existing blood vessels, which is observed both prenatally and postnatally. The placenta is an organ composed of a variety of fetal-derived cells, including ECFCs, and therefore has significant potential as a source of fetal ECFCs for tissue engineering. AIM: To investigate the possibility of isolating clonal ECFCs from human early gestation chorionic villi (CV-ECFCs) of the placenta, and assess their potential for tissue engineering. METHODS: The early gestation chorionic villus tissue was dissociated by enzyme digestion. Cells expressing CD31 were selected using magnetic-activated cell sorting, and plated in endothelial-specific growth medium. After 2-3 wks in culture, colonies displaying cobblestone-like morphology were manually picked using cloning cylinders. We characterized CV-ECFCs by flow cytometry, immunophenotyping, tube formation assay, and Dil-Ac-LDL uptake assay. Viral transduction of CV-ECFCs was performed using a Luciferase/tdTomato-containing lentiviral vector, and transduction efficiency was tested by fluorescent microscopy and flow cytometry. Compatibility of CV-ECFCs with a delivery vehicle was determined using an FDA approved, small intestinal submucosa extracellular matrix scaffold. RESULTS: After four passages in 6-8 wks of culture, we obtained a total number of 1.8 × 10(7) CV-ECFCs using 100 mg of early gestational chorionic villus tissue. Immunophenotypic analyses by flow cytometry demonstrated that CV-ECFCs highly expressed the endothelial markers CD31, CD144, CD146, CD105, CD309, only partially expressed CD34, and did not express CD45 and CD90. CV-ECFCs were capable of acetylated low-density lipoprotein uptake and tube formation, similar to cord blood-derived ECFCs (CB-ECFCs). CV-ECFCs can be transduced with a Luciferase/tdTomato-containing lentiviral vector at a transduction efficiency of 85.1%. Seeding CV-ECFCs on a small intestinal submucosa extracellular matrix scaffold confirmed that CV-ECFCs were compatible with the biomaterial scaffold. CONCLUSION: In summary, we established a magnetic sorting-assisted clonal isolation approach to derive CV-ECFCs. A substantial number of CV-ECFCs can be obtained within a short time frame, representing a promising novel source of ECFCs for fetal treatments.

Background Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cell-based gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells. Methods In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice. Results In vivo bioluminescence imaging showed that the ECFC only and the co-transplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks post-transplantation. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype, and generated functional vasculature. Next, co-transplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13 ± 19.84 μl to 155.78 ± 44.93 μl in a tail-clip assay. Conclusions This work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA. Electronic supplementary material The online version of this article (10.1186/s13287-019-1138-8) contains supplementary material, which is available to authorized users.

Table S1. (1) Primer sequence of RT-PCR. (2) Primer sequence of EndMT. (3) Primer sequence of Real-time PCR. Figure S1. Characterization of the transduced cord blood ECFCs. Figure S2. Characterization of the transduced PMSCs. Figure S3. Short-term monitoring of cell retention after transplantation in neonatal mice. (DOCX 786 kb)

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII ( FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach.

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cellbased gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells. In this study, we sought to determine whether co-transplantation of endothelial colonyforming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice. In vivo bioluminescence imaging showed that the ECFC only and the cotransplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks of age. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype and generated functional vasculature. Next, cotransplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13±19.84µl to 155.78±44.93µl in a tail-clip assay. This work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA. Funding Statement: This work was in part supported by UC Davis Department of Surgery departmental fund, and the UC Davis Medical Center Interdepartmental Seed Grant. Declaration of Interests: The authors declare: "None." Ethics Approval Statement: All animal procedures were approved by The University of California, Davis (UCD) institutional animal care and use committee (IACUC). All facilities used during the study period were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

Through traditional medicine, there were diseases and disorders that previously remained untreated or were simply thought to be incurable. Since the discovery of mesenchymal stem cells (MSCs), there has been a flurry of research to develop MSC-based therapy for diseases and disorders. It is now well-known that MSCs do not typically engraft after transplantation and exhibit their therapeutic effect via a paracrine mechanism. In addition to secretory proteins, MSCs also produce extracellular vesicles (EVs), membrane-bound nanovesicles containing proteins, DNA and RNA. The secreted vesicles then interact with target cells and deliver their contents, imparting their ultimate therapeutic effect. Unlike the widely studied cancer cells, the yield of MSC-exosomes is a limiting factor for large-scale production for cell-free therapies. Here we summarise potential approaches to increase the yield of such vesicles while maintaining or enhancing their efficacy by engineering the extracellular environment and intracellular components of MSCs.

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Both mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) have been used independently to correct the HA phenotype, but poor survival and insufficient retention of transplanted cells in the long term limit their application. In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) with placental mesenchymal stem cells (PMSCs) can achieve stable, long-term engraftment. To investigate stem cell engraftment and therapeutic potential, ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector. To distinguish between PMSCs and ECFCs in the co-transplantation group, a Td-Tomato containing lentiviral vector was used to label PMSCs while a green fluorescent protein (GFP) labeling vector was used to label ECFCs. They were then transplanted intramuscularly either as ECFC only group, PMSC only group or co-transplantation of ECFCs and PMSCs group into neonatal (3 days) or adult (12 weeks) immune deficient NOD scid gamma (NSG) mice. Using an in vivo bioluminescence imaging system, we investigated cell engraftment for up to 26 weeks after transplantation. ECFC only and the co-transplantation groups achieved better engraftment than PMSCs only group in both neonatal and adult mice during the entire study period. The co-transplantation group showed higher engraftment rate than the ECFC only group at 16 weeks and 20 weeks post-transplantation. In addition, transplantation of ECFCs only or in combination with PMSCs in neonatal period achieved higher engraftment than transplantation in adult period. Immunohistochemical analyses were further employed to determine the fate and functionality of the engrafted cells. We found that engrafted ECFCs maintained endothelial phenotype and expressed high level of FVIII at 26 weeks post-transplantation. The presence of ECFCs in the co-transplantation group also promoted formation of functional vasculature after transplantation. To study the functional reversal of HA phenotype we co-transplanted ECFCs and PMSCs into F8 knock-out HA mice. The transplanted cells reduced the blood loss volume from 562.13±19.84µl of blood to 155.78±44.93µl in a tail clip assay in these HA mice. This work demonstrates that co-transplantation of ECFCs with PMSCs at the neonatal period is a promising strategy to achieve stable, long-term engraftment of transplanted cells, and thus holds great promise for the treatment of HA and other genetic diseases and tissue regeneration applications. Disclosures No relevant conflicts of interest to declare.