Feasibility of large experimental animal models in testing novel therapeutic strategies for diabetes

Diabetes is among the top 10 causes of death in adults and caused approximately four million deaths worldwide in 2017.The incidence and prevalence of diabetes is predicted to increase.To alleviate this potentially severe situation,safer and more effective therapeutics are urgently required.Mice have long been the mainstay as preclinical models for basic research on diabetes,although they are not ideally suited for translating basic knowledge into clinical applications.To validate and optimize novel therapeutics for safe application in humans,an appropriate large animal model is needed.Large animals,especially pigs, are well suited for biomedical research and share many similarities with humans,including body size,anatomical features,physiology,and pathophysiology.Moreover,pigs already play an important role in translational studies,including clinical trials for xenotransplantation.Progress in genetic engineering over the past few decades has facilitated the development of transgenic animals,including porcine models of diabetes.This article discusses features that attest to the attractiveness of genetically modified porcine models of diabetes for testing novel treatment strategies using recent technical advances.

Myelin protein zero (P0)- and Wnt1-Cre marked muscle resident neural crest-derived mesenchymal progenitor cells give rise to heterotopic ossification in mouse models

Heterotopic ossification(HO)describes bone formation at non-skeletal sites and results from traumatic injury,surgery,or genetic disease such as fibrodysplasia ossificans progressiva(FOP).1,2 Although it is known that BMP signaling regulates HO,knowledge about the developmental origin of the osteogenic progenitors responsible for the BMP-associated metamorphosis is comparably less.With the use of transgenic mice and labelled neural crest-derived cell,3 we found myelin protein zero(P0,or MPZ)-and Wnt1-lineage cells give rise to BMP-7 induced adult ectopic cartilage and bone.

Early development and functional properties of tryptase/chymase double-positive mast cells from human pluripotent stem cells

Mast cells (MCs) play a pivotal role in the hypersensitivity reaction by regulating the innate and adaptive immune responses. Humans have two types of MCs. The first type, termed MCTC, is found in the skin and other connective tissues and expresses both tryptase and chymase, while the second, termed MCT, which only expresses tryptase, is found primarily in the mucosa. MCs induced from human adult-type CD34+ cells are reported to be of the MCT type, but the development of MCs during embryonic/fetal stages is largely unknown. Using an efficient coculture system, we identified that a CD34+c-kit+ cell population, which appeared prior to the emergence of CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs), stimulated robust production of pure Tryptase+Chymase+ MCs (MCTCs). Single-cell analysis revealed dual development directions of CD34+c-kit+ progenitors, with one lineage developing into erythro-myeloid progenitors (EMP) and the other lineage developing into HSPC. Interestingly, MCTCs derived from early CD34+c-kit+ cells exhibited strong histamine release and immune response functions. Particularly, robust release of IL-17 suggested that these early developing tissue-type MCTCs could play a central role in tumor immunity. These findings could help elucidate the mechanisms controlling early development of MCTCs and have significant therapeutic implications.

Semi-3D cultures using Laminin 221 as a coating material for human induced pluripotent stem cells

It was previously believed that human induced pluripotent stem cells(hiPSCs)did not show adhesion to the coating material Laminin 221,which is known to have specific affinity for cardiomyocytes.In this study,we report that human mononuclear cell-derived hiPSCs,established with Sendai virus vector,form peninsular-like colonies rather than embryonic stem cell-like colonies;these peninsular-like colonies can be passaged more than 10 times after establishment.Additionally,initializationdeficient cells with residual Sendai virus vector adhered to the coating material Laminin 511 but not to Laminin 221.Therefore,the expression of undifferentiated markers tended to be higher in hiPSCs established on Laminin 221 than on Laminin 511.On Laminin 221,hiPSCs15M66 showed a semi-floating colony morphology.The expression of various markers of cell polarity was significantly lower in hiPSCs cultured on Laminin 221 than in hiPSCs cultured on Laminin 511.Furthermore,201B7 and 15M66 hiPSCs showed 3D cardiomyocyte differentiation on Laminin 221.Thus,the coating material Laminin 221 provides semi-floating culture conditions for the establishment,culture and induced differentiation of hiPSCs.

Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm

RUNXI is absolutely required for definitive hematopoiesis, but the function of RUNXlb/c, two isoforms of human RUNX1, is unclear. We established inducible RUNXlb/c-overexpressing human embryonic stem cell （hESC） lines, in which RUNXlb/c overexpression prevented the emergence of CD34＋ cells from early stage, thereby drastically reducing the production of hematopoi- etic stem/prognnitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 ceU co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor （TGF）-β signaling pathway, indicating a close relationship between RUNX1b/c and TGF-β pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models.

BCR–ABL-specific CD4^(+) T-helper cells promote the priming of antigen-specific cytotoxic T cells via dendritic cells

The advent of tyrosine kinase inhibitor(TKI)therapy markedly improved the outcome of patients with chronic-phase chronic myeloid leukemia(CML).However,the poor prognosis of patients with advanced-phase CML and the lifelong dependency on TKIs are remaining challenges;therefore,an effective therapeutic has been sought.The BCR–ABL p210 fusion protein’s junction region represents a leukemia-specific neoantigen and is thus an attractive target for antigen-specific T-cell immunotherapy.BCR–ABL p210 fusion-region-specific CD4+T-helper(Th)cells possess antileukemic potential,but their function remains unclear.In this study,we established a BCR–ABL p210 b3a2 fusion-region-specific CD4+Th-cell clone(b3a2-specific Th clone)and examined its dendritic cell(DC)-mediated antileukemic potential.The b3a2-specific Th clone recognized the b3a2 peptide in the context of HLA-DRB1*09:01 and exhibited a Th1 profile.Activation of this clone through T-cell antigen receptor stimulation triggered DC maturation,as indicated by upregulated production of CD86 and IL-12p70 by DCs,which depended on CD40 ligation by CD40L expressed on b3a2-specific Th cells.Moreover,in the presence of HLA-A*24:02-restricted Wilms tumor 1(WT1)235–243 peptide,DCs conditioned by b3a2-specific Th cells efficiently stimulated the primary expansion of WTI-specific cytotoxic T lymphocytes(CTLs).The expanded CTLs were cytotoxic toward WT1235–243-peptide-loaded HLA-A*24:02-positive cell lines and exerted a potent antileukemic effect in vivo.However,the b3a2-specific Th-clone-mediated antileukemic CTL responses were strongly inhibited by both TKIs and interferon-α.Our findings indicate a crucial role of b3a2-specific Th cells in leukemia antigen-specific CTL-mediated immunity and provide an experimental basis for establishing novel CML immunotherapies.

Inhibition of aryl hydrocarbon receptor signaling promotes the terminal differentiation of human erythroblasts

The aryl hydrocarbon receptor(AHR)plays an important role during mammalian embryo development.Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells.AHR also regulates the functional maturation of blood cells,such as T cells and megakaryocytes.However,little is known about the role of AHR modulation during the development of erythroid cells.In this study,we used the AHR antagonist StemRegenin 1(SR1)and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin during different stages of human erythropoiesis to elucidate the function of AHR.We found that antagonizing AHR signaling improved the production of human embryonic stem cell derived erythrocytes and enhanced erythroid terminal differentiation.RNA sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes.We found that SR1 accelerated F-actin remodeling in terminally differentiated erythrocytes,favoring their maturation of the cytoskeleton and enucleation.We demonstrated that the effects of AHR inhibition on erythroid maturation were associated with F-actin remodeling.Our findings help uncover the mechanism for AHRmediated human erythroid cell differentiation.We also provide a new approach toward the large-scale production of functionally mature human pluripotent stem cell-derived erythrocytes for use in translational applications.