Kidney Capsule Of Mice Research Articles

3D-printed scaffolds with controlled-releasing compounds for ectopic activation of dormant ovarian follicles

Throughout a woman’s reproductive life, hundreds of functioning follicles are activated for development, while thousands of dormant follicles remain in the ovaries. Dormant follicle activation in vitro is an effective clinical strategy for women with fertility needs who suffer from ovarian dysfunction. However, activating dormant follicles in vivo is still a challenge due to difficulties such as delivering stimulators to local sites. Here, we developed a compound-preloaded microporous scaffold by combining three-dimensional (3D) printing techniques with pharmacological activators to support and stimulate the activation and development of primordial follicles. The gelatin/alginate composite scaffolds exhibited exceptional mechanical properties and biological compatibility, effectively supporting the survival of ovarian granulosa cells for more than 7 days, which is essential for oocyte development. Furthermore, the ovarian tissue-scaffold complex successfully survived after transplantation under the mouse kidney capsule, demonstrating its excellent biocompatibility. After pre-mixing widely used clinical activators into the bio-ink, the scaffold had the ability to gradually release the mixture of compounds, effectively activating primordial follicles. By transplanting the ovary-scaffold complex containing activators into the mouse abdominal subcutaneous pocket, dormant follicles could be activated subcutaneously and developed into growing follicles. The number of growing follicles is approximately three times higher compared to the group without activators. In conclusion, by integrating biomaterials, activators, and 3D printing technology, we have developed 3D-printed biological scaffolds that can ectopically support primordial follicle activation and development in vivo. This novel approach could provide a promising strategy for treating ovarian insufficiency and endocrine disorders in the clinic, without the need for in situ ovarian tissue grafting.

Modeling high-risk Wilms tumors enables the discovery of therapeutic vulnerability

Wilms tumor (WT) is the most common pediatric kidney cancer treated with standard chemotherapy. However, less-differentiated blastemal type of WT often relapses. To model the high-risk WT for therapeutic intervention, we introduce pluripotency factors into WiT49, a mixed-type WT cell line, to generate partially reprogrammed cells, namely WiT49-PRCs. When implanted into the kidney capsule in mice, WiT49-PRCs form kidney tumors and develop both liver and lung metastases, whereas WiT49 tumors do not metastasize. Histological characterization and gene expression signatures demonstrate that WiT49-PRCs recapitulate blastemal-predominant WTs. Moreover, drug screening in isogeneic WiT49 and WiT49-PRCs leads to the identification of epithelial- or blastemal-predominant WT-sensitive drugs, whose selectivity is validated in patient-derived xenografts (PDXs). Histone deacetylase (HDAC) inhibitors (e.g., panobinostat and romidepsin) are found universally effective across different WT and more potent than doxorubicin in PDXs. Taken together, WiT49-PRCs serve as a blastemal-predominant WT model for therapeutic intervention to treat patients with high-risk WT.

Generation of rat offspring from ovarian oocytes by xenotransplantation

The idea of utilizing unused oocytes present in the ovaries has been tested in various ways to produce offspring. However, only a limited number of studies succeeded in offspring generation. They include transplantation of ovaries into autologous or allogeneic animals, and acquisition of pups from oocytes obtained by transplanting mouse ovaries into immunodeficient rats. Here we report successful production of rat oocytes by transplanting rat ovaries under the kidney capsule of immunodeficient mice with addition of hormone administration to the mice. In addition, these oocytes were developed by in vitro fertilization, and transplanted into the oviducts of pseudopregnant rats, resulting in successful delivery of pups. The modified gene of the donor rat was confirmed to be correctly inherited to the pups. These results show that xenotransplantation of ovarian tissue makes it possible to leave offspring, beginning a new phase in developmental engineering.

Thymic microenvironment's impact on immunosenescence.

Age-related thymic involution is characterized by the loss of T cell development and the supporting epithelial network, which are replaced by adipose tissue. We previously showed that aging functionally impairs lymphohematopoietic progenitor cells, including thymic early T cell progenitors (ETPs), contributing to thymic involution. Considering that the thymic microenvironment is essential for thymocyte incubation, we aimed to investigate its role in age-related thymic involution and the mechanisms underlying these changes. The challenge in studying these processes led us to transplant T cell-depleted fetal thymus tissue into the kidney capsule of aged mice. This model allowed us to identify the mechanisms driving age-related changes in the thymic microenvironment and to assess whether these changes could be reversed. Flow cytometry was used to detect naïve T cells (CD62L+CD44-), including CD4 CD8 double-negative, double-positive, and single-positive T cells. Real-time PCR was used to detect and quantify signal-joint T cell receptor excision circles. We rearranged δRec-ΨJα in murine peripheral blood leukocytes to evaluate the thymic output of newly developed naïve T cells in the mice and gene expression in the thymus. Age-related thymic involution decreased naïve T cells and increased memory T cells, while fetal thymus transplantation improved thymic output and T cell production and reversed the impairment of thymopoiesis due to thymic involution in aged mice. Furthermore, the expression of key cytokines was restored and ETPs in the aged mice showed normal thymic T cell development. Our study suggests that degenerative changes in the thymic microenvironment are the primary cause of thymic dysfunction, leading to immunosenescence associated with age-related thymic involution.

Skeletal Myoblast Cells Enhance the Function of Transplanted Islets in Diabetic Mice.

Islet transplantation (ITx) is an established and safe alternative to pancreas transplantation for type 1 diabetes mellitus (T1DM) patients. However, most ITx recipients lose insulin independence by 3 years after ITx due to early graft loss, such that multiple donors are required to achieve insulin independence. In the present study, we investigated whether skeletal myoblast cells could be beneficial for promoting angiogenesis and maintaining the differentiated phenotypes of islets. In vitro experiments showed that the myoblast cells secreted angiogenesis-related cytokines (vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and stromal-derived factor-1α (SDF-1α)), contributed to maintenance of differentiated islet phenotypes, and enhanced islet cell insulin secretion capacity. To verify these findings in vivo, we transplanted islets alone or with myoblast cells under the kidney capsule of streptozotocin-induced diabetic mice. Compared with islets alone, the group bearing islets with myoblast cells had a significantly lower average blood glucose level. Histological examination revealed that transplants with islets plus myoblast cells were associated with a significantly larger insulin-positive area and significantly higher number of CD31-positive microvessels compared to islets alone. Furthermore, islets cotransplanted with myoblast cells showed JAK-STAT signaling activation. Our results suggest two possible mechanisms underlying enhancement of islet graft function with myoblast cells cotransplantation: "indirect effects" mediated by angiogenesis and "direct effects" of myoblast cells on islets via the JAK-STAT cascade. Overall, these findings suggest that skeletal myoblast cells enhance the function of transplanted islets, implying clinical potential for a novel ITx procedure involving myoblast cells for patients with diabetes.

RAAS-deficient organoids indicate delayed angiogenesis as a possible cause for autosomal recessive renal tubular dysgenesis

Autosomal Recessive Renal Tubular Dysgenesis (AR-RTD) is a fatal genetic disorder characterized by complete absence or severe depletion of proximal tubules (PT) in patients harboring pathogenic variants in genes involved in the Renin–Angiotensin–Aldosterone System. To uncover the pathomechanism of AR-RTD, differentiation of ACE-/- and AGTR1-/- induced pluripotent stem cells (iPSCs) and AR-RTD patient-derived iPSCs into kidney organoids is leveraged. Comprehensive marker analyses show that both mutant and control organoids generate indistinguishable PT in vitro under normoxic (21% O2) or hypoxic (2% O2) conditions. Fully differentiated (d24) AGTR1-/- and control organoids transplanted under the kidney capsule of immunodeficient mice engraft and mature well, as do renal vesicle stage (d14) control organoids. By contrast, d14 AGTR1-/- organoids fail to engraft due to insufficient pro-angiogenic VEGF-A expression. Notably, growth under hypoxic conditions induces VEGF-A expression and rescues engraftment of AGTR1-/- organoids at d14, as does ectopic expression of VEGF-A. We propose that PT dysgenesis in AR-RTD is primarily a non-autonomous consequence of delayed angiogenesis, starving PT at a critical time in their development.

Characterisation of transgenic pigs expressing a human T cell-depleting anti-CD2 monoclonal antibody.

Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell-mediated rejection in baboons. Local expression of a depleting anti-CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock-in transgenic pigs expressing the chimeric anti-CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock-in pigs in which MHCIP was replaced by the β-cell-specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines. A PIP-diliximab knock-in construct was prepared and validated by transfection of NIT-1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock-in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP-diliximab and PIP-diliximab knock-in pigs was characterised at the mRNA and protein levels using RT-qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP-diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice. NIT-1 cells stably transfected with the PIP-diliximab knock-in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in β cells. PIP-diliximab knock-in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP-diliximab pigs, but was not detectable in PIP-diliximab pigs. Likewise, diliximab was present in the serum of MHCIP-diliximab pigs, at a mean concentration of 1.8 μg/mL, but was not detected in PIP-diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP-diliximab pigs but not of PIP-diliximab pigs. Whole genome sequencing (WGS) of a PIP-diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock-in clone used to generate the PIP-diliximab pigs. Islet xenografts from neonatal MHCIP-diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ. Diliximab was widely expressed in MHCIP-diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP-diliximab pigs is sufficient to prevent T cell-mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP-diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock-in cells used for SCNT.

Magnetic resonance imaging of pancreatic islets using tissue–adhesive particles containing iron oxide nanoparticles

Post–transplantation tracking of pancreatic islets is a prerequisite for advancing cell therapy to treat type 1 diabetes. Magnetic resonance imaging (MRI) has emerged as a safe and non–invasive technique for visualizing cells in clinical applications. In this study, we proposed a novel MRI contrast agent formulation by encapsulating iron oxide nanoparticles (IONPs) in poly(lactic–co–glycolic acid) (PLGA) particles functionalized with a tissue adhesive polydopamine (PD) layer (IONP–PLGA–PD MS). Intriguingly, our particles facilitated efficient and robust labeling through a one–step process, allowing for the incorporation of a substantial amount of IONPs without detrimental impacts on the viability and functionality of pancreatic islets. The MRI signals emanating from islets labeled using our particles were found to be stable over 30 days in vitro and 60 days when transplanted under kidney capsules of diabetic mice. These results suggest that our approach provides a potential platform for monitoring the fate of pancreatic islets after transplantation.

MRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells.

Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by invitro culture or invivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLCs. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development.

Atp6i deficient mouse model uncovers transforming growth factor-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation

The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood. We found that Atp6i deficient mice (Atp6i−/−) arrested tooth root formation, indicated by truncated Hertwig’s epithelial root sheath (HERS) progression. Furthermore, Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation. Atp6i−/− mice had largely decreased expression of odontoblast differentiation marker gene expression profiles (Col1a1, Nfic, Dspp, and Osx) in the alveolar bone. Atp6i−/− mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers. Additionally, there was a significant reduction in Smad2/3 activation, inhibiting transforming growth factor-β (TGF-β) signaling in Atp6i−/− odontoblasts. Through treating pulp precursor cells with Atp6i−/− or wild-type OC bone resorption-conditioned medium, we found the latter medium to promote odontoblast differentiation, as shown by increased odontoblast differentiation marker genes expression (Nfic, Dspp, Osx, and Runx2). This increased expression was significantly blocked by anti-TGF-β1 antibody neutralization, whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i−/− OC conditioned medium. Importantly, ectopic TGF-β1 partially rescued root development and root dentin deposition of Atp6i−/− mice tooth germs were transplanted under mouse kidney capsules. Collectively, our novel data shows that the prevention of TGF-β1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation. As such, this study uncovers TGF-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.

The mTOR Inhibitor Rapamycin Counteracts Follicle Activation Induced by Ovarian Cryopreservation in Murine Transplantation Models.

Background and Objectives: Ovarian tissue cryopreservation followed by autotransplantation (OTCTP) is currently the only fertility preservation option for prepubertal patients. Once in remission, the autotransplantation of frozen/thawed tissue is performed when patients want to conceive. A major issue of the procedure is follicular loss directly after grafting mainly due to follicle activation. To improve follicular survival during the OTCTP procedure, we inhibited the mTOR pathway involved in follicle activation using rapamycin, an mTOR inhibitor. Next, we compared two different in vivo models of transplantation: the recently described non-invasive heterotopic transplantation model between the skin layers of the ears, and the more conventional and invasive transplantation under the kidney capsule. Materials and Methods: To study the effects of adding rapamycin during cryopreservation, 4-week-old C57BL/6 mouse ovaries, either fresh, slow-frozen, or slow-frozen with rapamycin, were autotransplanted under the kidney capsule of mice and recovered three weeks later for immunohistochemical (IHC) analysis. To compare the ear with the kidney capsule transplantation model, fresh 4-week-old C57BL/6 mouse ovaries were autotransplanted to either site, followed by an injection of either LY294002, a PI3K inhibitor, vehicle control, or neither, and these were recovered three weeks later for IHC analysis. Results: Rapamycin counteracts cryopreservation-induced follicle proliferation, as well as AKT and mTOR pathway activation, in ovaries autotransplanted for three weeks under the kidney capsule of mice. Analyses of follicle proliferation, mTOR activation, and the effects of LY294002 treatment were similar in transplanted ovaries using either the ear or kidney capsule transplantation model. Conclusions: By adding rapamycin during the OTCTP procedure, we were able to transiently maintain primordial follicles in a quiescent state. This is a promising method for improving the longevity of the ovarian graft. Furthermore, both the ear and kidney capsule transplantation models were suitable for investigating follicle activation and proliferation and pharmacological strategies.

Greb1 Transiently Accelerates Pancreatic β-Cell Proliferation in Diabetic Mice Exposed to Estradiol

Decrease of pancreatic β cells leads to diabetes. In an inducible cAMP early suppressor (ICER-Iγ) transgenic mouse model of severe type 2 diabetes with reduced insulin production and depleted β cells, supplementation with high concentrations of 17β-estradiol (E2) markedly enhances β-cell proliferation and normalizes glucose levels. The current study explored the underlying mechanisms leading to a dynamic increase of β cells and pathologic changes in diabetic mice exposed to E2. Gene expression profiling of pancreatic islets of 6-month-old ICER-transgenic mice recovering from diabetes due to elevated E2 levels identified growth regulation by estrogen in breast cancer 1 (Greb1) as a gene significantly up-regulated during the recovery phase. To substantiate this, β-cell-specific Greb1-deficient mice were generated, and Greb1 was shown to be essential for recovery of depleted β cells in diabetic mice. Graft growth and glucose lowering were observed in 50 islets with increased Greb1 expression transplanted adjacent to E2 pellets beneath the kidney capsule of streptozotocin-induced diabetic mice. Greb1 expression due to a drastic increase in exogenous or endogenous E2 was transient and closely correlated with changes in E2-related and some cell cycle-related genes. These findings provide new insights into invivo proliferation of deficient β cells and suggest the possibility of new therapeutic approaches targeting pancreatic β cells that could revolutionize the concept of diabetes treatment, which has been considered difficult to cure completely.

Direct Differentiation of Bone Marrow Mononucleated Cells Into Insulin-Producing Cells Using 4 Specific Soluble Factors.

Bone marrow-derived stem cells are self-renewing and multipotent adult stem cells that differentiate into several types of cells. Here, we investigated a unique combination of 4 differentiation-inducing factors (DIFs), including putrescine (Put), glucosamine (GlcN), nicotinamide, and BP-1-102, to develop a differentiation method for inducing mature insulin-producing cells (IPCs) and apply this method to bone marrow mononucleated cells (BMNCs) isolated from mice. BMNCs, primed with the 4 soluble DIFs, were differentiated into functional IPCs. BMNCs cultured under the defined conditions synergistically expressed multiple genes, including those for PDX1, NKX6.1, MAFA, NEUROG3, GLUT2, and insulin, related to pancreatic beta cell development and function. They produced insulin/C-peptide and PDX1, as assessed using immunofluorescence and flow cytometry. The induced cells secreted insulin in a glucose-responsive manner, similar to normal pancreatic beta cells. Grafting BMNC-derived IPCs under kidney capsules of mice with streptozotocin (STZ)-induced diabetes alleviated hyperglycemia by lowering blood glucose levels, enhancing glucose tolerance, and improving glucose-stimulated insulin secretion. Insulin- and PDX1-expressing cells were observed in the IPC-bearing graft sections of nephrectomized mice. Therefore, this study provides a simple protocol for BMNC differentiation, which can be a novel approach for cell-based therapy in diabetes mellitus.

1729-P: Role of ß-Cell Senescence in Islet Transplantation

Islet transplantation (Tx) is an effective intervention to prevent severe hypoglycemia and provide insulin independence in type 1 diabetes (T1D). Previous studies have shown that islets from older donors are less effective in reversing blood glucose (BG) than those of younger ones. Cellular senescence leads to age-related β-cell dysfunction and drives alloimmune responses to organs from older donors, but can be targeted with senolysis. However, the mechanism of pancreatic β-cell senescence in transplant grafts and its pathological significance are unknown. Therefore, we performed two types of islet Tx to test the hypothesis that cellular senescence may play a role in loss of graft functionality/viability and that the use of senolysis may ameliorate it: 1) overnight cultured 600 IEQ islets from normoglycemic rats were transplanted into kidney capsule of streptozotocin-induced diabetic immunodeficient mice (Stz-Ms). After 2 weeks, the grafts were removed and compared with the cultured islets (control). In Stz-Ms, the nuclear exclusion rate of Hmgb1 and p21-positive cells tended to increase, and the mRNA levels of p16, Il-6, and β-cells related genes were significantly higher than in control islets indicating β-cell senescence after transplantation. To test if the elimination of p16 positive senescent β-cells from transplanted islets improved BG levels, 2) 440 IEQ islets from INK-ATTAC mice (p16Ink4a mediated apoptosis through targeted activation of caspase) were transplanted into Stz-Ms and treated intraperitoneally with BB Homodimerizer (BB) or vehicle for 2 terms of 3 consecutive days, of which 2 weeks of rest. With senolysis, there was a decrease in fed glucose levels and p16 mRNA expression while expression of hallmark β-cell genes increased by 74%, which correlates with improved response to high glucose. In conclusion, islet transplantation increases β-cell senescence in grafts and senolysis improves β-cell function. This has important implications in increasing graft function and survival in the treatment of T1D. Disclosure K.Iwasaki: None. P.Carapeto: None. C.Aguayo-mazzucato: None. Funding Manpei Suzuki Diabetes Foundation

231-LB: Prolactin Modulates the Differentiation of Pancreatic Ductal Cell to Islet-Like Cell

Objective: Pancreatic ductal cells will be differentiated into islet-like cell with the activation of PRL-PRLR signaling pathway in vitro. Methods: Primary mouse and human pancreatic ductal cells were obtained by CK-19 labelling and MACS sorting of digested pancreatic tissue, and selected cells were cultured with recombinant mouse prolactin and recombinant human prolactin. Subsequently, the cell clusters formed by the pancreatic ductal cells were stained by anti-insulin, anti-GCK, anti-GLUT2, anti-PC1/3 Abs. In vitro, glucose stimulated insulin secretion (GSIS) was quantified. Prolactin related signaling pathway, glucose transportation and insulin secretion related proteins were assessed by immunoblotting. Lastly, the in vitro formed islet-like cells were transplanted under the kidney capsule of diabetic mice. To assess the role for blood glucose regulation of transplanted islet-like cells, blood glucose, c-peptide and intravenous glucose tolerance tests were performed and immunofluorescence staining for insulin and glucagon was performed on the grafts. Results: At day 7, the pancreatic ductal cells, also called progenitor cells, expressed CK-19; at day 14, the ductal cells were aggregated and formed sphere-shaped cell clusters. At day 21, the cell clusters stained positive for insulin and etc. By in vitro GSIS test, we measured a 2.58±0.32-fold increase of insulin secretion in response to a 16.7 mM glucose stimulation. Immunoblotting demonstrated that PDX-1, MafA and other genes were expressed. At day 21, the cell clusters expressed PDX-1, insulin and glucagon. In vivo studies showed that diabetic mice transplanted with islet-like cells, maintained blood glucose levels below 300 mg/dL with c-peptide detected in the serum. Further tissue staining indicated that the transplanted islet-like cells contained both β-cells and α-cells. Conclusion: Out study shows that in vitro stimulation by prolactin might differentiate pancreatic ductal cells into islet-like cells. Disclosure Y. Wang: None. Funding National Natural Science Foundation of China (81802504); Sichuan Science and Technology Bureau (2022YFH0005, 2023YFH0010)

Tissue engineering human thymus from iPSCs to support human T cell development

Abstract Thymus is the primary immune organ responsible for the generation of diverse and self-tolerant T cells that are essential for fighting off invading pathogens while maintaining immune homeostasis. Loss of thymus function, due to either genetic mutations or environmental insults, can lead to prolonged immunodeficiency that can be life-threatening. Rejuvenating the function of the thymus is challenging as the organ undergoes irreversible involution at young age, during which thymic epithelial cells (TECs) – the thymic stromal cells key to the education and selection of developing T cells – are progressively replaced by infiltrating adipocytes. Regeneration of thymus from the patient’s own stem cells will have tremendous clinical benefits in treating immunodeficiency and immunodysregulatory disorders. We reported here the tissue-engineering of functional artificial thymus from inducible pluripotent stem cells (iPSCs). Taking advantage of the 3D alginate encapsulation technology, we developed a novel protocol to induce the differentiation and maturation of human iPSCs into TECs (iPSC-TECs) at high efficiency. To bioengineer the thymus, iPSC-TECs were injected into decellularized murine thymus scaffolds, in conjunction with T cell progenitors derived from hematopoietic stem/progenitor cells (HSPCs) harvested from the umbilical cord blood. When transplanted under the kidney capsules of hematopoietic humanized mice (aka immunodeficient mice engrafted with HSPCs), iPSC-TEC thymus can support developed a diverse population of human T cells that could mediate adaptive immune responses. Our findings suggest that functional human thymus can be generated from patient-derived iPSCs for regenerative medicine. Supported in part by the NIH (R01 AI123392 and R21 AI126335), NSF (CBET 1804728), and Highmark Health Award (A023948-HIGHMARK-LANNI-FAN).

Maturation of Nephrons by Implanting hPSC-derived Kidney Progenitors Under Kidney Capsules of Unilaterally Nephrectomized Mice.

Human pluripotent stem cell (hPSC)-derived kidney organoids may contribute to disease modeling and the generation of kidney replacement tissues. However, the realization of such applications requires the induction of hPSCs into functional mature organoids. One of the key questions for this process is whether a specific vascular system exists for nephrogenesis. Our previous study showed that short-term (2 weeks) implantation of hPSC-derived organoids below the kidney capsules of unilaterally nephrectomized and immunodeficient mice resulted in the enlargement of organoids and production of vascular cells, although signs of maturation were lacking. Organoids were induced for 15 days in vitro and then grafted below kidney capsules of the same unilaterally nephrectomized immunodeficient mouse model to examine whether medium-term (4 weeks) implantation could improve organoid maturation and vascularization, as evaluated by immunofluorescence and transmission electron microscopy. We demonstrated that after 2-4 weeks of implantation, renal organoids formed host-derived vascularization and matured without any exogenous vascular endothelial growth factor. Glomerular filtration barrier maturation was evidenced by glomerular basement membrane deposition, perforated glomerular endothelial cell development, and apical, basal podocyte polarization. A polarized monolayer epithelium and extensive brush border were also observed for tubular epithelial cells. Our results indicate that the in vivo microenvironment is important for the maturation of human kidney organoids. Stromal expansion and a reduction of nephron structures were observed following longer-term (12 weeks) implantation, suggesting effects on off-target cells during the induction process. Accordingly, induction efficiency and transplantation models should be improved in the future.

In vivo development of immune tissue in human intestinal organoids transplanted into humanized mice

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal–immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.

Effects of human induced pluripotent stem cell-derived intestinal organoids on colitis-model mice

IntroductionUlcerative colitis (UC) is an inflammatory bowel disease characterized by repeated remissions and relapses. Immunosuppressive drugs have facilitated the induction and maintenance of remission in many patients with UC. However, immunosuppressive drugs cannot directly repair impaired intestinal mucosa and are insufficient for preventing relapse. Therefore, new treatment approaches to repair the damaged epithelium in UC have been attempted through the transplantation of intestinal organoids, which can be differentiated into mucosa by embedding in Matrigel, generated from patient-derived intestinal stem cells. The method, however, poses the challenge of yielding sufficient cells for UC therapy, and patient-derived cells might already have acquired pathological changes. In contrast, human induced pluripotent stem (iPS) cells generated from healthy individuals are infinitely proliferated and can be differentiated into target cells. Recently developed human iPS cell-derived intestinal organoids (HIOs) aim to generate organoids that closely resemble the adult intestine. However, no study till date has reported HIOs injected into in vivo inflammatory models, and it remains unclear whether HIOs with cells that closely resemble the adult intestine or with intestinal stem cells retain the better ability to repair tissue in colitis.MethodsWe generated two types of HIOs via suspension culture with and without small-molecule compounds: HIOs that include predominantly more intestinal stem cells [HIO (A)] and those that include predominantly more intestinal epithelial and secretory cells [HIO (B)]. We examined whether the generated HIOs engrafted in vivo and compared their ability to accelerate recovery of the damaged tissue.ResultsFindings showed that the HIOs expressed intestinal-specific markers such as caudal-type homeobox 2 (CDX2) and villin, and HIOs engrafted under the kidney capsules of mice. We then injected HIOs into colitis-model mice and found that the weight and clinical score of the mice injected with HIO (A) recovered earlier than that of the mice in the sham group. Further, the production of mucus and the expression of cell proliferation markers and tight junction proteins in the colon tissues of the HIO (A) group were restored to levels similar to those observed in healthy mice. However, neither HIO (A) nor HIO (B) could be engrafted into the colon.ConclusionsEffective cell therapy should directly repair tissue by engraftment at the site of injury. However, the difference in organoid property impacting the rate of tissue repair in transplantation without engraftment observed in the current study should be considered a critical consideration in the development of regenerative medicine using iPS-derived organoids.

P12.11: Immunomodulatory Microporous Annealed Particle (MAP) Gel Protects Delivery of Islets to Promote Islet Transplantation Outcomes

Introduction: To investigate if coating dissociated or whole islets with microporous annealed particle (MAP) gel could support cell function and viability and improve islet transplantation outcomes. Methods: a) C57BL/6 mouse islets were mixed with MAP gel and cultured in 24 well plates at 37°C. The viability of islets was scored after Propidium Iodide-Fluorescein Diacetate (PI/FDA) staining and Glucose stimulated insulin secretion (GSIS) assay performed on Day 1, 3, 5, and 7 post-culture. The experiment was done thrice. b) Single cells from C57BL/6 mouse dissociated islets were mixed with MAP gel, and cultured in 24 well plates at 37°C. Viability of cells was scored after PI/FDA staining and GSIS assay performed 1, 24, 48, and 72 hours post-culture. c) 100 C57BL/6 mouse islets were dissociated into single cells by Trypsin, mixed with MAP gel, and transplanted into syngeneic diabetic recipients under the kidney capsule. Blood glucose (BG) was measured daily. Results: Mouse islets protected with MAP gel displayed similar GSIS and viability scores when compared to uncoated islets. Both groups retained above 95% viability, with the stimulation index (SI) of MAP gel-protected islets 3.2 and that of uncoated islets is 2.4. Viability of dissociated islets protected with MAP gel was 84% with a SI=8.5 at 72 hours post culture. In contrast, uncoated dissociated islets were nearly dead with only 35% viability and an SI=0.38 at 72 hours post culturing. In MAP scaffold, dissociated islet cell viability was density dependent − cell viability in the 50 and 12.5 dissociated islet conditions was on average >78%, while dissociated cells from 2.5 islets demonstrated 56% viability after 72 hours. In in vivo studies, no significant difference was observed in the ability to return Streptozotocin-induced diabetic mice to normoglycemia when either MAP gel coated or uncoated whole islets (75-100 islets) were transplanted under the mouse kidney capsule. C57BL/6 mouse islets (100 islets) that were dissociated to single cells and co-transplanted with MAP gel under the kidney capsule, permanently returned transplanted mice to normoglycemia, with glucose levels below 200 mg/dL within 18 days post-transplantation (n=6). In contrast, uncoated dissociated islets failed to restore normoglycemia in tranplanted mice. Treatment efficacy continued 40 days post-transplantation (time mice were monitored). The average blood glucose measured at Day 30 was 163 ± 38.2 mg/dL. Recipient mice transplanted with cells from dissociated islets without MAP gel remained hyperglycemic (n=6/group). Conclusion: The MAP scaffold protects delivery of dissociated islet cells, and helps promote islet transplantation outcomes. Moreover, MAP scaffold provides a possible platform for delivering stem cell-derived insulin-secreting cells. LaunchPad Ignite. NIH grant: NIBIB R21 Trailblazer [1R21EB028971-01A1].