Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation ...non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.
The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative ...for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5+ population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1+TNNT2+ intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations.
Display omitted
•Comprehensive gene expression profiles on human cardiogenesis are reported•LGR5 is identified as a key regulator on human-specific cono-ventriculogenesis•LGR5 signaling may be associated with certain human congenital heart diseases
Sahara et al. performed population and single-cell transcriptional analysis of in vitro cardiac differentiation from human embryonic stem cells and human embryonic/fetal hearts. They identified human-specific cardiogenic programs driven by LGR5, which has an important role in cono-ventricular region development in human cardiogenesis.
Long segmental repair of trachea stenosis is an intractable condition in the clinic. The reconstruction of an artificial substitute by tissue engineering is a promising approach to solve this unmet ...clinical need. 3D printing technology provides an infinite possibility for engineering a trachea. Here, we 3D printed a biodegradable reticular polycaprolactone (PCL) scaffold with similar morphology to the whole segment of rabbits' native trachea. The 3D-printed scaffold was suspended in culture with chondrocytes for 2 (Group I) or 4 (Group II) weeks, respectively. This in vitro suspension produced a more successful reconstruction of a tissue-engineered trachea (TET), which enhanced the overall support function of the replaced tracheal segment. After implantation of the chondrocyte-treated scaffold into the subcutaneous tissue of nude mice, the TET presented properties of mature cartilage tissue. To further evaluate the feasibility of repairing whole segment tracheal defects, replacement surgery of rabbits' native trachea by TET was performed. Following postoperative care, mean survival time in Group I was 14.38 ± 5.42 days, and in Group II was 22.58 ± 16.10 days, with the longest survival time being 10 weeks in Group II. In conclusion, we demonstrate the feasibility of repairing whole segment tracheal defects with 3D printed TET.
Cell-based therapies offer an exciting and novel treatment for heart repair following myocardial infarction (MI). However, these therapies often suffer from poor cell viability and engraftment rates, ...which involve many factors, including the hypoxic conditions of the infarct environment. Meanwhile, vascular endothelial growth factor (VEGF) has previously been employed as a therapeutic agent to limit myocardial damage and simultaneously induce neovascularization. This study took an approach to transiently overexpress VEGF protein, in a controlled manner, by transfecting human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with VEGF mRNA prior to transplantation. The conditioning of iPSC-CMs with VEGF mRNA ultimately led to greater survival rates of the transplanted cells, which promoted a stable vascular network in the grafted region. Furthermore, bulk RNA transcriptomics data and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) and AGE-RAGE signaling pathways were significantly upregulated in the VEGF-treated iPSC-CMs group. The over-expression of VEGF from iPSC-CMs stimulated cell proliferation and partially attenuated the hypoxic environment in the infarcted area, resulting in reduced ventricular remodeling. This study provides a valuable solution for the survival of transplanted cells in tissue-engineered heart regeneration and may further promote the application of modified mRNA (modRNA) in the field of tissue engineering.
Display omitted
Fu et al. describe a study combining novel mRNA technology with cell-based therapy to enhance cardiac regeneration. This study provides a reliable solution for increasing the survival rates of transplanted cells in cardiac muscle and may further promote the application of modified mRNA in the field of tissue engineering.
Major cardiovascular events including myocardial infarction (MI) continue to dominate morbidity rates in the developed world. Although multiple device therapies and various pharmacological agents ...have been shown to improve patient care and reduce mortality rates, clinicians and researchers alike still lack a true panacea to regenerate damaged cardiac tissue. Over the previous two to three decades, cardiovascular stem cell therapies have held great promise. Several stem cell-based approaches have now been shown to improve ventricular function and are documented in preclinical animal models as well as phase I and phase II clinical trials. More recently, the cardiac progenitor cell has begun to gain momentum as an ideal candidate for stem cell therapy in heart disease. Here, we will highlight the most recent advances in cardiac stem/progenitor cell biology in regard to both the basics and applied settings.
Bone has a remarkable potential for self-healing and repair, yet several injury types are non-healing even after surgical or non-surgical treatment. Regenerative therapies that induce bone repair or ...improve the rate of recovery are being intensely investigated. Here, we probed the potential of bone marrow stem cells (BMSCs) engineered with chemically modified mRNAs (modRNA) encoding the hBMP-2 and VEGF-A gene to therapeutically heal bone. Induction of osteogenesis from modRNA-treated BMSCs was confirmed by expression profiles of osteogenic related markers and the presence of mineralization deposits. To test for therapeutic efficacy, a collagen scaffold inoculated with modRNA-treated BMSCs was explored in an in vivo skull defect model. We show that hBMP-2 and VEGF-A modRNAs synergistically drive osteogenic and angiogenic programs resulting in superior healing properties. This study exploits chemically modified mRNAs, together with biomaterials, as a potential approach for the clinical treatment of bone injury and defects.
Cardiogenic growth factors play important roles in heart development. Placental growth factor (PLGF) has previously been reported to have angiogenic effects; however, its potential role in ...cardiogenesis has not yet been determined. We analyze single-cell RNA-sequencing data derived from human and primate embryonic hearts and find PLGF shows a biphasic expression pattern, as it is expressed specifically on ISL1
second heart field progenitors at an earlier stage and on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) at later stages. Using chemically modified mRNAs (modRNAs), we generate a panel of cardiogenic growth factors and test their effects on enhancing cardiomyocyte (CM) and EC induction during different stages of human embryonic stem cell (hESC) differentiations. We discover that only the application of PLGF modRNA at early time points of hESC-CM differentiation can increase both CM and EC production. Conversely, genetic deletion of PLGF reduces generation of CMs, SMCs and ECs in vitro. We also confirm in vivo beneficial effects of PLGF modRNA for development of human heart progenitor-derived cardiac muscle grafts on murine kidney capsules. Further, we identify the previously unrecognized PLGF-related transcriptional networks driven by EOMES and SOX17. These results shed light on the dual cardiomyogenic and vasculogenic effects of PLGF during heart development.
Fat grafting, as a standard treatment for numerous soft tissue defects, remains unpredictable and technique-dependent. Human adipose-derived stem cells (hADSCs) are promising candidates for ...cell-assisted therapy to improve graft survival. As free-living fat requires nutritional and respiratory sources to thrive, insufficient and unstable vascularization still impedes hADSC-assisted therapy. Recently, cytotherapy combined with modified mRNA (modRNA) encoding vascular endothelial growth factor (VEGF) has been applied for the treatment of ischemia-related diseases. Herein, we hypothesized that VEGF modRNA (modVEGF)-engineered hADSCs could robustly enhance fat survival in a fat graft transplantation model.
hADSCs were acquired from lipoaspiration and transfected with modRNAs. Transfection efficiency and expression kinetics of modRNAs in hADSCs were first evaluated in vitro. Next, we applied an in vivo Matrigel plug assay to assess the viability and angiogenic potential of modVEGF-engineered hADSCs at 1 week post-implantation. Finally, modVEGF-engineered hADSCs were co-transplanted with human fat in a murine model to analyze the survival rate, re-vascularization, proliferation, fibrosis, apoptosis, and necrosis of fat grafts over long-term follow-up.
Transfections of modVEGF in hADSCs were highly tolerable as the modVEGF-engineered hADSCs facilitated burst-like protein production of VEGF in both our in vitro and in vivo models. modVEGF-engineered hADSCs induced increased levels of cellular proliferation and proangiogenesis when compared to untreated hADSCs in both ex vivo and in vivo assays. In a fat graft transplantation model, we provided evidence that modVEGF-engineered hADSCs promote the optimal potency to preserve adipocytes, especially in the long-term post-transplantation phase. Detailed histological analysis of fat grafts harvested at 15, 30, and 90 days following in vivo grafting suggested the release of VEGF protein from modVEGF-engineered hADSCs significantly improved neo-angiogenesis, vascular maturity, and cell proliferation. The modVEGF-engineered hADSCs also significantly mitigated the presence of fibrosis, apoptosis, and necrosis of grafts when compared to the control groups. Moreover, modVEGF-engineered hADSCs promoted graft survival and cell differentiation abilities, which also induced an increase in vessel formation and the number of surviving adipocytes after transplantation.
This current study demonstrates the employment of modVEGF-engineered hADSCs as an advanced alternative to the clinical treatment involving soft-tissue reconstruction and rejuvenation.
The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein ...remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics.
A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing.
Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes.
Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.
PDF
