hESC-derived Cells Research Articles

FORMATION OF MALIGNANT, METASTATIC SMALL CELL LUNG CANCERS THROUGH OVERPRODUCTION OF cMYC PROTEIN IN TP53 AND RB1 DEPLETED PULMONARY NEUROENDOCRINE CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS.

We recently described our initial efforts to develop a model for small cell lung cancer (SCLC) derived from human embryonic stem cells (hESCs) that were differentiated to form pulmonary neuroendocrine cells (PNECs), a putative cell of origin for neuroendocrine-positive SCLC. Although reduced expression of the tumor suppressor genes TP53 and RB1 allowed the induced PNECs to form subcutaneous growths in immune-deficient mice, the tumors did not display the aggressive characteristics of SCLC seen in human patients. Here we report that the additional, doxycycline-regulated expression of a transgene encoding wild-type or mutant cMYC protein promotes rapid growth, invasion, and metastasis of these hESC-derived cells after injection into the renal capsule. Similar to others, we find that the addition of cMYC encourages the formation of the SCLC-N subtype, marked by high levels of NEUROD1 RNA. Using paired primary and metastatic samples for RNA sequencing, we observe that the subtype of SCLC does not change upon metastatic spread and that production of NEUROD1 is maintained. We also describe histological features of these malignant, SCLC-like tumors derived from hESCs and discuss potential uses of this model in efforts to control and better understand this recalcitrant neoplasm.

Promotion of Epithelial Healing in Oral Mucositis by hESC-Derived Mesenchymal Stem Cells via the PI3K/AKT Pathway

Introduction: Oral mucositis (OM) is a common and debilitating side effect of cancer therapies such as radiotherapy, chemotherapy, hematopoietic cell transplant, or their combinations. Method: This study focused on the reparative effects of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) in OM and possible mechanisms. An ulcer model was created in the rat buccal mucosa to mimic an in vivo animal model of OM mucosal injury, and hESC-MSCs were injected 48h later to assess their reparative effects. In vitro, the efficacy of hESC-MSCs in regulating apoptosis and proliferation in LPS- or 5-fluorouracil (5-FU)-injured HaCaT cells was studied using a transwell coculture system. Subsequently, the PI3K inhibitor LY24002 was used to assess whether hESC-MSCs regulated injured HaCaT cells through the PI3K/AKT pathway. In vivo, we found that hESC-MSCs injection promoted OM healing in rats through the acceleration of re-epithelialization and a decrease in apoptosis. Results: In vitro, our findings revealed that the hESC-MSCs treatment led to a reduction in the quantity of HaCaT cells undergoing apoptosis. Western blot analysis revealed that hESC-MSCs activated AKT, resulting in increased protein levels of PCNA and BCL-2 and decreased protein levels of Bax and Caspase-3 whereas LY294002 reversed these changes. Conclusion: These findings suggest that hESC-MSCs promoted OM wound healing by stimulating the proliferation of epithelial cells and inhibiting their apoptosis in rat models. Furthermore, hESC-MSCs might mediate the PI3K/AKT pathway to modulate apoptosis/proliferation injured by LPS or 5-FU in HaCaT cells.

Expandable hESC-derived cardiovascular progenitor cells generate functional cardiac lineage cells for microtissue construction

BackgroundCardiovascular progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) are considered valuable cell sources for investigating cardiovascular physiology in vitro. Meeting the diverse needs of this application requires the large-scale production of CPCs in an in vitro environment. This study aimed to use an effective culture system utilizing signaling factors for the large-scale expansion of hESC-derived CPCs with the potential to differentiate into functional cardiac lineage cells.Methods and resultsInitially, CPCs were generated from hESCs using a 4-day differentiation protocol with a combination of four small molecules (CHIR99021, IWP2, SB-431542, and purmorphamine). These CPCs were then expanded and maintained in a medium containing three factors (bFGF, CHIR, and A83-01), resulting in a > 6,000-fold increase after 8 passages. These CPCs were successfully cryopreserved for an extended period in late passages. The expanded CPCs maintained their gene and protein expression signatures as well as their differentiation capacity through eight passages. Additionally, these CPCs could differentiate into four types of cardiac lineage cells: cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts, demonstrating appropriate functionality. Furthermore, the coculture of these CPC-derived cardiovascular lineage cells in rat tail collagen resulted in cardiac microtissue formation, highlighting the potential of this 3D platform for studying cardiovascular physiology in vitro.ConclusionIn conclusion, expandable hESC-derived CPCs demonstrated the ability to self-renewal and differentiation into functional cardiovascular lineage cells consistently across passages, which may apply as potential cell sources for in vitro cardiovascular studies.Graphical

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 μg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

Directed differentiation of human embryonic stem cells into conjunctival epithelial cells

Severe conjunctival damage can lead to extensive ocular cicatrisation, fornix shortening, and even ocular surface failure, resulting in significant vision impairment. Conjunctival reconstruction is the primary therapeutic strategy for these clinical conjunctival diseases. However, there have been limited studies on induced differentiation of conjunctival epithelial cells derived from stem cells. In this study, we established a chemical defined differentiation protocol from human embryonic stem cells (hESCs) into conjunctival epithelial cells. hES cell line H1 was used for differentiation, and RT-qPCR, immunofluorescence staining, Periodic-acid-Schiff staining (PAS), and transcriptome analysis were employed to identify the differentiated cells. Here, to imitate the development of the vertebrate conjunctiva, hESCs were induced using a three-step process involving first chetomin was used to induce ocular surface ectoderm, then nicotinamide was used to induce ocular surface epithelial progenitor cells, and finally epidermal growth factor, keratinocyte growth factor and other factors were used to differentiate mature conjunctival epithelial cells. hESC-derived conjunctival epithelial cells expressed mature conjunctival epithelial lineage markers (including PAX6, P63, K13). The presence of goblet cells was confirmed by positive PAS. Transcriptome analysis revealed that hESC-derived conjunctival epithelial cells possessed a more naïve phenotype, and exhibited greater proliferation capacity compared to mature human conjunctival epithelial cells, suggesting their potential as alternative seed cells for conjunctival reconstruction.

Chimerization of human ESC-derived extraembryonic cells with the mouse blastocyst.

It has been reported that human embryonic stem cells (hESCs) treated with BMP4 and inhibitors of TGFβ signaling (A83-01) and FGF signaling (PD173074), called BAP, can efficiently differentiate to extraembryonic (ExE) cells in vitro. Due to restricted access to human embryos, it is ethically impossible to test the developmental potential of ExE cells in vivo. Here, we demonstrate that most ExE cells expressed molecular markers for both trophoblasts (TBs) and amniotic cells (ACs). Following intra-uterine transplantation, ExE cells contributed to the mouse placenta. More interestingly, ExE cells could chimerize with the mouse blastocyst as, after injection into the blastocyst, they penetrated its trophectoderm. After implantation of the injected blastocysts into surrogate mice, human cells were found at E14 in placental labyrinth, junction zones, and even near the uterine decidua, expressed placental markers, and secreted human chorionic gonadotropin. Surprisingly, ExE cells also contributed to cartilages of the chimeric embryo with some expressing the chondrogenic marker SOX9, consistent with the mesodermal potential of TBs and ACs in the placenta. Deleting MSX2, a mesodermal determinant, restricted the contribution of ExE cells to the placenta. Thus, we conclude that hESC-derived ExE cells can chimerize with the mouse blastocyst and contribute to both the placenta and cartilages of the chimera consistent with their heteogenious nature. Intra-uterus and intra-blastocyst injections are novel and sensitive methods to study the developmental potential of ExE cells.

Identification of RNA-based cell-type markers for stem-cell manufacturing systems with a statistical scoring function

Cell-type biomarkers are useful in stem-cell manufacturing to monitor cell purity, quantity, and quality. However, the study on cell-type markers, specifically for stem cell manufacture, is limited. Emerging questions include which RNA transcripts can serve as biomarkers during stem cell culture and how to discover these biomarkers efficiently and precisely. We developed a scoring function system to identify RNA biomarkers with RNA-seq data for systems that have a limited number of cell types. We applied the method to two data sets, one for extracellular RNAs (ex-RNAs) and the other for intracellular microRNAs (miRNAs). The first data set has RNA-seq data of ex-RNAs from cell culture media for six different types of cells, including human embryonic stem cells. To get the RNA-seq data from intracellular miRNAs, we cultured three types of cells: human embryonic stem cells (H9), neural stem cells (NSC), hESC-derived endothelial cells (EC) and conducted small RNA-seq to their intracellular miRNAs. Using these data, we identified a set of ex-RNAs/smRNAs as candidates of biomarkers for different types of cells for cell manufacture. The validity of these findings was confirmed by the utilization of additional data sets and experimental procedures. We also used deep-learning-based prediction methods and simulated data to validate these discovered biomarkers.

BACH1 regulates the differentiation of vascular smooth muscle cells from human embryonic stem cells via CARM1-mediated methylation of H3R17

The role of BACH1 in the process of vascular smooth muscle cell (VSMC) differentiation from human embryonic stem cells (hESCs) remains unknown. Here, we find that the loss of BACH1 in hESCs attenuates the expression of VSMC marker genes, whereas overexpression of BACH1 after mesoderm induction increases the expression of VSMC markers during invitro hESC-VSMC differentiation. Mechanistically, BACH1 binds directly to coactivator-associated arginine methyltransferase 1 (CARM1) during invitro hESC-VSMC differentiation, and this interaction is mediated by the BACH1 bZIP domain. BACH1 recruits CARM1 to VSMC marker gene promoters and promotes VSMC marker expression by increasing H3R17me2 modification, thus facilitating invitro VSMC differentiation from hESCs after the mesoderm induction. The increased expression of VSMC marker genes by BACH1 overexpression is partially abolished by inhibition of CARM1 or the H3R17me2 inhibitor TBBD in hESC-derived cells. These findings highlight the critical role of BACH1 in hESC differentiation into VSMCs by CARM1-mediated methylation of H3R17.

Aligned skeletal muscle assembly on a biofunctionalized plant leaf scaffold.

Decellularized plant scaffolds have drawn attention as alternative tissue culture platforms due to their wide accessibility, biocompatibility, and diversity of innate microstructures. Particularly, in this work, monocot leaves with innate uniaxial micropatterned topography were utilized to promote cell alignment and elongation. The leaf scaffold was biofunctionalized with poly(PEGMEMA-r-VDM-r-GMA) copolymer that prevented non-specific protein adsorption and was modified with cell adhesive RGD peptide to enable cell adhesion and growth in serum-free media. The biofunctionalized leaf supported the adhesion, growth, and alignment of various human cells including embryonic stem cells (hESC) derived muscle cells. The hESC-derived myogenic progenitor cells cultured on the biofunctionalized leaf scaffold adopted a parallel orientation and were elongated along the leaf topography. These cells showed significant early myogenic differentiation and muscle-like bundled myotube formation. The aligned cells formed compact myotube assemblies and showed uniaxial muscle contraction under chemical stimulation, a critical requirement for developing functional skeletal muscle tissue. Polymer-functionalized plant leaf scaffolds offer a novel human cell culture platform and have potential in human tissue engineering applications that require parallel alignment of cells. STATEMENT OF SIGNIFICANCE: Plant scaffolds are plentiful sources in nature and present a prefabricated construct to present topographical cues to cells. Their feature width is ideal for human cell alignment and elongation, especially for muscle cells. However, plant scaffolds lack proteins that support mammalian cell culture. We have developed a polymer coated leaf scaffold that enables cell adhesion and growth in serum-free media. Human muscle cells cultured on the biofunctionalized leaf, aligned along the natural parallel micro-patterned leaf topography, and formed muscle-like bundled myotube assemblies. These assemblies showed uniaxial muscular contraction, a critical requirement for developing functional skeletal muscle tissue. The biodiversity of the plant materials offers a novel human cell culture platform with potential in human tissue engineering.

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice.

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

ZnT8 loss-of-function accelerates functional maturation of hESC-derived beta cells and resists metabolic stress in diabetes

ZnT8 loss-of-function accelerates functional maturation of hESC-derived beta cells and resists metabolic stress in diabetes

Ex vivo cultivated retinal pigment epithelial cell transplantation for the treatment of rabbit corneal endothelial dysfunction

ObjectiveStem cell therapy is a promising strategy for the treatment of corneal endothelial dysfunction, and the need to find functional alternative seed cells of corneal endothelial cells (CECs) is urgent. Here, we determined the feasibility of using the retinal pigment epithelium (RPE) as an equivalent substitute for the treatment of corneal endothelial dysfunction.MethodsRPE cells and CECs in situ were obtained from healthy New Zealand male rabbits, and the similarities and differences between them were analyzed by electron microscopy, immunofluorescent staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Rabbit primary RPE cells and CECs were isolated and cultivated ex vivo, and Na+/K+-ATPase activity and cellular permeability were detected at passage 2. The injection of cultivated rabbit primary RPE cells, CECs and human embryonic stem cell (hESC)-derived RPE cells was performed on rabbits with corneal endothelial dysfunction. Then, the therapeutic effects were evaluated by corneal transparency, central corneal thickness, enzyme linked immunosorbent assay (ELISA), qRT-PCR and immunofluorescent staining.ResultsThe rabbit RPE cells were similar in form to CECs in situ and ex vivo, showing a larger regular hexagonal shape and a lower cell density, with numerous tightly formed cell junctions and hemidesmosomes. Moreover, RPE cells presented a stronger barrier and ionic pumping capacity than CECs. When intracamerally injected into the rabbits, the transplanted primary RPE cells could dissolve corneal edema and decrease corneal thickness, with effects similar to those of CECs. In addition, the transplantation of hESC-derived RPE cells exhibited a similar therapeutic effect and restored corneal transparency and thickness within seven days. qRT-PCR results showed that the expressions of CEC markers, like CD200 and S100A4, increased, and the RPE markers OTX2, BEST1 and MITF significantly decreased in the transplanted RPE cells. Furthermore, we have demonstrated that rabbits transplanted with hESC-derived RPE cells maintained normal corneal thickness and exhibited slight pigmentation in the central cornea one month after surgery. Immunostaining results showed that the HuNu-positive transplanted cells survived and expressed ZO1, ATP1A1 and MITF.ConclusionRPE cells and CECs showed high structural and functional similarities in barrier and pump characteristics. Intracameral injection of primary RPE cells and hESC-derived RPE cells can effectively restore rabbit corneal clarity and thickness and maintain normal corneal function. This study is the first to report the effectiveness of RPE cells for corneal endothelial dysfunction, suggesting the feasibility of hESC-derived RPE cells as an equivalent substitute for CECs.

HESC-Derived Epicardial Cells Promote Repair of Infarcted Hearts in Mouse and Swine.

Myocardial infarction (MI) causes excessive damage to the myocardium, including the epicardium. However, whether pluripotent stem cell-derived epicardial cells (EPs) can be a therapeutic approach for infarcted hearts remains unclear. Here, the authors report that intramyocardial injection of human embryonic stem cell-derived EPs (hEPs) at the acute phase of MI ameliorates functional worsening and scar formation in mouse hearts, concomitantly with enhanced cardiomyocyte survival, angiogenesis, and lymphangiogenesis. Mechanistically, hEPs suppress MI-induced infiltration and cytokine-release of inflammatory cells and promote reparative macrophage polarization. These effects are blocked by a type I interferon (IFN-I) receptor agonist RO8191. Moreover, intelectin 1 (ITLN1), abundantly secreted by hEPs, interacts with IFN-β and mimics the effects of hEP-conditioned medium in suppression of IFN-β-stimulated responses in macrophages and promotion of reparative macrophage polarization, whereas ITLN1 downregulation in hEPs cancels beneficial effects of hEPs in anti-inflammation, IFN-I response inhibition, and cardiac repair. Further, similar beneficial effects of hEPs are observed in a clinically relevant porcine model of reperfused MI, with no increases in the risk of hepatic, renal, and cardiac toxicity. Collectively, this study reveals hEPs as an inflammatory modulator in promoting infarct healing via a paracrine mechanism and provides a new therapeutic approach for infarcted hearts.

348-OR: Differentiation of Adult Islet Cell Precursors into ß- and α-like Cells for Islet Transplantation

Advances in protocols to convert human embryonic and inducible pluripotent stem cells (hESCs and iPSCs) into pancreatic β cells has allowed us to generate islet cell organoids in culture for potential use in islet cell transplantation. Here, we demonstrate that dedifferentiated β cells and/or NGN-/RGS16+ islet cell precursors (ICPs) can be harvested from adult pancreatic tissue and converted into β- and α-like cells. In this study, we used ICPs derived from COBE bag pancreatic tissue remnants from organ donors to produce islet cell organoids capable of releasing both insulin and glucagon in response to high (16.7 mM) and low (2.8 mM) glucose conditions, respectively. These organoids displayed zinc-containing granule staining by dithizone and exhibited GSIS with stimulation indices comparable to human islets. ICPs produced genetic profiles similar to hESC-derived endocrine progenitor cells at stages 5-7 (S5-7). Using small molecule ISX9 and GSK/DYRK inhibitors GNF and harmine to induce and activate calcineurin (CN)/NFAT signaling, we were able to expand and differentiate ICPs into β- and α-like cells. CN/NFAT upregulated genes that promote cell cycle and proliferation (S5) and islet cell differentiation genes (S5-7). Islet cell type fate and function could be modulated by hedgehog/GLI signaling. Whereas introduction of sonic hedgehog (Shh) inhibitor SANT-1 at S5 favored production of β-like cells, Shh signaling agonists promoted differentiation to α-like cells up to S7. Overall, these results indicate that adult ICPs can be harvested from donor tissue and expanded for differentiation into functional β- and α-like cells. The capability to expand and differentiate adult ICPs could potentially provide an unlimited supply of islet organoid tissue for islet cell transplantation. Disclosure C.Darden: None. J.Kuncha: None. J.D.Mattke: None. B.Naziruddin: None. M.C.Lawrence: None. Funding Baylor Scott & White Health

In Vivo Tumorigenicity of the 20q11.21 Amplicon in an Engraftment Model of hPSCs and Differentiated Liver Cells.

Human pluripotent stem cells (hPSCs) are a promising source of somatic cells for clinical applications and disease modelling. However, during culture they accumulate genetic aberrations such as amplification of 20q11.21 which occurs in approximately 20% of extensively cultured hPSC lines and confers a BCL2L1-mediated survival advantage. During the production of the large number of cells required for transplantation and therapy these aberrations may become unavoidable which has important safety implications for therapies and may also impact upon disease modelling. Presently, these risks are poorly understood; whilst it is apparent that large-scale genetic aberrations can pose an oncogenic risk, the risks associated with smaller, more insidious changes have not been fully explored. In this report, the effects of engraftment of human embryonic stem cells (hESCs) and hESC-derived hepatocyte-like cells (HLCs) with and without amplification of the 20q11.21 minimal amplicon and isochromosome 20q (i20q) in SCID-beige mice are presented. The cells were tracked in vivo using a luminescent reporter over a period of approximately four months. Intrasplenic injection of hESCs showed greater engraftment potential and the formation of more severely disruptive lesions in the liver and spleen of animals injected with cells containing 20q11.21 compared with i20q and wild type. HLCs with 20q11.21 engrafted more successfully and formed more severely disruptive lesions than wild type cells or cells with i20q. These results reinforce the notion that karyotyping of therapeutic hPSC is required for transplant, and suggest that screening for known common aberrations is necessary. Further work to identify commonly arising genetic aberrations should be performed and routine screening for hPSCs intended for therapeutic use should be used.

Genome editing HLA alleles for a pilot immunocompatible hESC line in a Chinese hESC bank for cell therapies

Robust allogeneic immune reactions after transplantation impede the translational pace of human embryonic stem cells (hESCs)-based therapies. Selective genetic editing of human leucocyte antigen (HLA) molecules has been proposed to generate hESCs with immunocompatibility, which, however, has not been specifically designed for the Chinese population yet. Herein, we explored the possibility of customizing immunocompatible hESCs based on Chinese HLA typing characteristics. We generated an immunocompatible hESC line by disrupting HLA-B, HLA-C, and CIITA genes while retaining HLA-A*11:01 (HLA-A*11:01-retained, HLA-A11R ), which covers ~21% of the Chinese population. The immunocompatibility of HLA-A11R hESCs was verified by in vitro co-culture and confirmed in humanized mice with established human immunity. Moreover, we precisely knocked an inducible caspase-9 suicide cassette into HLA-A11R hESCs (iC9-HLA-A11R ) to promote safety. Compared with wide-type hESCs, HLA-A11R hESC-derived endothelial cells elicited much weaker immune responses to human HLA-A11+ T cells, while maintaining HLA-I molecule-mediated inhibitory signals to natural killer (NK) cells. Additionally, iC9-HLA-A11R hESCs could be induced to undergo apoptosis efficiently by AP1903. Both cell lines displayed genomic integrity and low risks of off-target effects. In conclusion,we customized a pilot immunocompatible hESC cell line based on Chinese HLA typing characteristics with safety insurance. This approach provides a basis for establishment of a universal HLA-AR bank of hESCs covering broad populations worldwide and may speed up the clinical application of hESC-based therapies.

Evaluating the Potential for ABO-incompatible Islet Transplantation: Expression of ABH Antigens on Human Pancreata, Isolated Islets, and Embryonic Stem Cell-derived Islets.

ABO-incompatible transplantation has improved accessibility of kidney, heart, and liver transplantation. Pancreatic islet transplantation continues to be ABO-matched, yet ABH antigen expression within isolated human islets or novel human embryonic stem cell (hESC)-derived islets remain uncharacterized. We evaluated ABH glycans within human pancreata, isolated islets, hESC-derived pancreatic progenitors, and the ensuing in vivo mature islets following kidney subcapsular transplantation in rats. Analyses include fluorescence immunohistochemistry and single-cell analysis using flow cytometry. Within the pancreas, endocrine and ductal cells do not express ABH antigens. Conversely, pancreatic acinar tissues strongly express these antigens. Acinar tissues are present in a substantial portion of cells within islet preparations obtained for clinical transplantation. The hESC-derived pancreatic progenitors and their ensuing in vivo-matured islet-like clusters do not express ABH antigens. Clinical pancreatic islet transplantation should remain ABO-matched because of contaminant acinar tissue within islet preparations that express ABH glycans. Alternatively, hESC-derived pancreatic progenitors and the resulting in vivo-matured hESC-derived islets do not express ABH antigens. These findings introduce the potential for ABO-incompatible cell replacement treatment and offer evidence to support scalability of hESC-derived cell therapies in type 1 diabetes.

The contribution of donated human embryos suitable for the production of embryonic stem cells to increase the quality of life: Selection and preparation of embryos in the Czech Republic.

Human embryonic stem cells (hESCs) have the unique ability to differentiate into any cell type in the human body and to proliferate indefinitely. Cell therapies involving hESC have shown very promising results for the treatment of certain diseases and confirmed the safety of hESC-derived cells for humans. They are used in cell therapy, mainly in targeted therapy of diseases that are currently incurable. The aim of this study was the derivation of clinical-grade hESCs usable in drug development, non-native medicine and cell therapy. Embryos were thawed, cultivated to the blastocyst stage if necessary, and assisted hatching was subsequently performed. Embryoblasts were mechanically isolated using narrow needles. Each line was kept as a separate batch. The derived hESCs were cultured under hypoxic culture conditions (5% O2, 5% CO2, 37°C) in a NutriStem® hPSC XF Medium with a daily medium change. From January 2018 to July 2020, 138 selected clients were asked for consent to donate embryos, of whom 52 did not respond, 19 terminated the storage of their embryos and 29 extended the storage. Only 38 clients (27.5%) agreed to donate embryos for the derivation of hESCs. At the same time, personal communication with clients took place and another 17 embryo donors were recruited. A total of 160 embryos from 55 donors aged 26-42 years were collected. The embryos were frozen at the blastocyst (33.1%) or morula (46.3%) stage. After the preparation of 64 embryos, embryoblasts were isolated and cultured. Finally, 7 hESC lines were obtained, 4 research-grade and 3 clinical-grade, the first in the Czech Republic. We established a current good manufacturing practice (cGMP)-defined xeno-free and feeder-free system for the derivation, culture and banking of clinical-grade hESC lines that are suitable for preclinical and clinical trials. The quality control testing with criteria concerning sterility, safety and characterization according to cGMP ensured the clinical-grade quality of hESC lines.

Transcriptome profiles associated with human periodontal ligament differentiation

ObjectivesTissue differentiation is regulated by transcription factors. This study aimed to identify candidate transcription factors that induce periodontal ligament (PDL) cell differentiation in human pluripotent stem cells (hPSCs). MethodsHuman PDL tissues were scraped from the root surfaces of extracted teeth for orthodontic treatment and cultured using the explant culture method. We used RNA-seq to generate gene expression profiles of third-passage PDL cells and compared them with those of undifferentiated human induced pluripotent stem cells (hiPSCs) and human embryonic stem cell (hESC)-derived neural crest (NC) cells (publicly available data). ResultsPrimary cultured PDL cells exhibited a spindle-shaped fibroblast-like appearance and the gene expression of several PDL cell-specific markers. The gene expression profiles of PDL cells were relatively similar to those of hESC-derived NC cells but not those of undifferentiated hiPSCs. Thirty-seven transcription factors were identified as upregulated genes in PDL cells. Pathway analysis showed that differentially expressed genes were enriched in several functional groups and pathways, including the SMAD 2/3 nuclear pathway. ConclusionsWe identified 37 upregulated transcription genes in primary cultured PDL cells compared with hESC-derived NC cells. Regulating these genes and the SMAD signaling pathway may be promising ways to induce PDL cells from hPSC-derived NC cells.

STRA6 is essential for induction of vascular smooth muscle lineages in human embryonic cardiac outflow tract development.

Retinoic acid (RA) signalling is essential for heart development, and dysregulation of the RA signalling can cause several types of cardiac outflow tract (OFT) defects, the most frequent congenital heart disease (CHD) in humans. Matthew-Wood syndrome is caused by inactivating mutations of a transmembrane protein gene STRA6 that transports vitamin A (retinol) from extracellular into intracellular spaces. This syndrome shows a broad spectrum of malformations including CHD, although murine Stra6-null neonates did not exhibit overt heart defects. Thus, the detailed mechanisms by which STRA6 mutations could lead to cardiac malformations in humans remain unclear. Here, we investigated the role of STRA6 in the context of human cardiogenesis and CHD. To gain molecular signatures in species-specific cardiac development, we first compared single-cell RNA sequencing (RNA-seq) datasets, uniquely obtained from human and murine embryonic hearts. We found that while STRA6 mRNA was much less frequently expressed in murine embryonic heart cells derived from the Mesp1+ lineage tracing mice (Mesp1Cre/+; Rosa26tdTomato), it was expressed predominantly in the OFT region-specific heart progenitors in human developing hearts. Next, we revealed that STRA6-knockout human embryonic stem cells (hESCs) could differentiate into cardiomyocytes similarly to wild-type hESCs, but could not differentiate properly into mesodermal nor neural crest cell-derived smooth muscle cells (SMCs) in vitro. This is supported by the population RNA-seq data showing down-regulation of the SMC-related genes in the STRA6-knockout hESC-derived cells. Further, through machinery assays, we identified the previously unrecognized interaction between RA nuclear receptors RARα/RXRα and TBX1, an OFT-specific cardiogenic transcription factor, which would likely act downstream to STRA6-mediated RA signalling in human cardiogenesis. Our study highlights the critical role of human-specific STRA6 progenitors for proper induction of vascular SMCs that is essential for normal OFT formation. Thus, these results shed light on novel and human-specific CHD mechanisms, driven by STRA6 mutations.