Regeneration of kidney tissue using in vitro cultured fetal kidney cells

Transplanting fetal kidney cells (FKCs) can regenerate kidney. This requires in vitro expansion in cell number to acquire enough cells for transplantation. However, FKCs may change their cellular characteristics during expansion and, thus, may not regenerate kidney tissue upon transplantation. We investigated how cell culture period affects cellular characteristics and in vivo regenerative potential of FKCs. As the passage number increased, cell growth rate and colony forming ability decreased while senescence and apoptosis increased. To examine in vivo regenerative potential, FKCs cultured through different numbers of passages were implanted into the parenchyma of kidneys of immunodeficient mice using fibrin gel for 4 wk. Histological analyses showed passage-dependent kidney tissue regeneration, and the regeneration was better when cells from lower number of passages were implanted. This result shows that in vitro culture of FKCs significantly affects the cell characteristics and in vivo tissue regenerative potential.     

Transplantation of betacellulin-transduced islets improves glucose intolerance in diabetic mice

Type 1 diabetes is an autoimmune disease caused by permanent destruction of insulin-producing pancreatic β cells and requires lifelong exogenous insulin therapy. Recently, islet transplantation has been developed, and although there have been significant advances, this approach is not widely used clinically due to the poor survival rate of the engrafted islets. We hypothesized that improving survival of engrafted islets through ex vivo genetic engineering could be a novel strategy for successful islet transplantation. We transduced islets with adenoviruses expressing betacellulin, an epidermal growth factor receptor ligand, which promotes β-cell growth and differentiation, and transplanted these islets under the renal capsule of streptozotocin-induced diabetic mice. Transplantation with betacellulin-transduced islets resulted in prolonged normoglycemia and improved glucose tolerance compared with those of control virus-transduced islets. In addition, increased microvascular density was evident in the implanted islets, concomitant with increased endothelial von Willebrand factor immunoreactivity. Finally, cultured islets transduced with betacellulin displayed increased proliferation, reduced apoptosis and enhanced glucose-stimulated insulin secretion in the presence of cytokines. These experiments suggest that transplantation with betacellulin-transduced islets extends islet survival and preserves functional islet mass, leading to a therapeutic benefit in type 1 diabetes.     

Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice

Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microg/microgL cDNA and 25 microgL effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.     

Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space

Stem and progenitor cells can be combined with polymer substrates to generate tissue equivalents in culture. The replacement of retinal tissue lost to disease or trauma using retinal progenitor cells (RPCs) delivered on polymer scaffolds and transplanted into the sub-retinal space of the damaged retina is a promising therapeutic strategy. Micromachining-based, ultra-thin PMMA poly(methyl methacrylate) scaffolds may provide a suitable cytoarchitectural environment for tissue engineering and transplantation to the diseased eye. Here, adhesion of RPCs to polymer, as well as migration and differentiation in the host retina were compared for PMMA scaffolds (6 microm thickness) with either smooth or porous (11 microm diameter) surface topography. RPCs were cultured under identical conditions on smooth or porous laminin-coated polymer scaffolds and transplanted into the subretinal space of C57BL/6 mice. RPCs could be cultured on both scaffolds with similar results, although transplantation with non-porous scaffolds showed limited RPC retention. Porous scaffolds demonstrated enhanced RPC adherence during transplantation and allowed for greater process outgrowth and cell migration into the host retinal layers. Integrated cells expressed the mature neuronal marker neurofilament-200 (nf-200), the glial marker glial fibrillary acidic protein (GFAP) and the retinal-specific marker recoverin. No host foreign body response was seen. In conclusion, ultra-thin film PMMA scaffolds micromachined to contain through pores retain adherent RPCs to a considerably greater extent than unmachined versions during the transplantation process and can serve as a biocompatible substrate for cell delivery in vivo.     

Fecal microbiota transplantation ameliorates atherosclerosis in mice with C1q/TNF-related protein 9 genetic deficiency

Despite the strong influence of the gut microbiota on atherosclerosis, a causal relationship between atherosclerosis pathophysiology and gut microbiota is still unverified. This study was performed to determine the impact of the gut microbiota on the pathogenesis of atherosclerosis caused by genetic deficiency. To elucidate the influence of the gut microbiota on atherosclerosis pathogenesis, an atherosclerosis-prone mouse model (C1q/TNF-related protein 9-knockout (CTRP9-KO) mice) was generated. The gut microbial compositions of CTRP9-KO and WT control mice were compared. Fecal microbiota transplantation (FMT) was performed to confirm the association between gut microbial composition and the progression of atherosclerosis. FMT largely affected the gut microbiota in both CTRP9-KO and WT mice, and all transplanted mice acquired the gut microbiotas of the donor mice. Atherosclerotic lesions in the carotid arteries were decreased in transplanted CTRP9-KO mice compared to CTRP9-KO mice prior to transplantation. Conversely, WT mice transplanted with the gut microbiotas of CTRP9-KO mice showed the opposite effect as that of CTRP9-KO mice transplanted with the gut microbiotas of WT mice. Here, we show that CTRP9 gene deficiency is related to the distribution of the gut microbiota in subjects with atherosclerosis. Transplantation of WT microbiotas into CTRP9-KO mice protected against the progression of atherosclerosis. Conversely, the transplantation of CTRP9-KO microbiotas into WT mice promoted the progression of atherosclerosis. Treating atherosclerosis by restoring gut microbial homeostasis may be an effective therapeutic strategy.Subject terms: Atherosclerosis, Experimental models of disease, Mechanisms of disease     

Effect of a scaffold fabricated thermally from acetylated PLGA on the formation of engineered cartilage

A MHDS has been employed to fabricate 3D scaffolds from PLGA with acetyl endgroups to achieve in vivo regeneration of cartilage tissue. The fabricated acetylated-PLGA scaffold showed open pores and interconnected structures. Rabbit chondrocytes were seeded on the PLGA scaffolds and transplanted immediately into subcutaneous sites of athymic mice. Chondrocytes transplantation with untreated PLGA scaffolds served as a control. Histological analysis of the implants at 4 weeks with H&E staining and alcian blue staining revealed higher extracellular matrix and GAG expression at the neocartilage in the PLGA-6Ac scaffolds than that of the PLGA-6OH scaffold group. This endgroup-modified scaffold may be useful for successful cartilage tissue engineering in orthopedic applications.     

Stromal Vascular Fraction Loaded Silk Fibroin Mats Effectively Support the Survival of Diabetic Mice after Pancreatic Islet Transplantation

The aim of this study is to assess whether stromal vascular fraction (SVF)‐soaked silk fibroin nonwoven mats (silk‐SVF) can preserve the functionality of encapsulated pancreatic endocrine cells (alginate‐PECs) after transplantation in the subcutaneous tissue of diabetic mice. Silk scaffolds are selected to create an effective 3D microenvironment for SVF delivery in the subcutaneous tissue before diabetes induction: silk‐SVF is subcutaneously implanted in the dorsal area of five healthy animals; after 15 d, mice are treated with streptozotocin to induce diabetes and then alginate‐PECs are implanted on the silk‐SVF. All animals appear in good health, increasing weight during time, and among them, one presents euglycemia until the end of experiments. On the contrary, when PECs are simultaneously implanted with SVF after diabetes induction, mice are euthanized due to suffering. This work clearly demonstrates that silk‐SVF creates a functional niche in subcutaneous tissue and preserves endocrine cell survival and engraftment.     

Migration of human neural stem cells toward an intracranial glioma

Many in vivo and in vitro studies have demonstrated the targeted migration of neural stem cells (NSC) to infiltrating brain tumors, including malignant glioma, highlighting a potential therapeutic approach. However, there is not enough information to apply this approach to clinical therapy. The most important things in stem cell therapy for brain tumors involve selecting the appropriate neural progenitor type and optimizing the efficiency of the cell engraftment. By histological analysis using two different live-dyes, human NSCs were shown to migrate away from the transplanted site in the direction of the expanding C6 glioma and to intermix with the tumor bed, especially with the tumor core. This intermixing occurred within 7 days when NSCs were implanted into glioma model. The time course of migratory HB1.F5 with the greatest mobility of three NSC lines was as follows. As early as 3 days after transplantation, several NSCs were found leaving the implant site, primarily approaching microsatellites and frontier cells located near the site of NSC implantation. Through 7 days post-transplantation, massive numbers of NSCs continued to be attracted to and interspersed with C6 glioma, and were finally distributed extensively throughout the whole tumor bed, including the core and penumbra of the tumor mass. However, NSCs appeared to penetrate into the tumor mass very well, whereas normal fibroblast cells could not migrate. These findings strengthen the potential for human NSCs as attractive vehicles to improve therapeutic gene delivery to cancer or glioma if they are optimized to selectively kill neoplastic cells.     

Paradoxical effects of elastase inhibitor guamerin on the tissue repair of two different wound models: sealed cutaneous and exposed tongue wounds

Innate elastase inhibitors are known to be putatively involved in the regulation of tissue inflammation by inhibiting polymorphonuclear leukocyte (PMN) derived proteinases. The aim of this study was to evaluate affects of leukocyte elastase suppression and PMN infiltration on wound healing in mouse by administering the recombinant elastase inhibitor guamerin (rEIG) in two different wound models; 1) impaired pin-punctured dorsal mucosa of anterior tongue wound, 60 mice, treated with saline containing rEIG that were fed ad libitum and 2) stable linear excisional cutaneous wound, 40 mice, covered with fibrin sealant containing rEIG. The progress of healing was analyzed by histological methods. The tongue wounds treated with rEIG became edematous around the pin-punctured tongue wound, and influx of inflammatory cells and PMN into the underlying stromal tissue were seen rapidly after wounding and peaked between 2-4 days. Whereas the control mice showed almost no wheal formation in the pin-punctured wound, a far lesser levels of PMN infiltration, and almost complete wound closure in 4 days. In the other model, the liner excisional cutaneous wound treated with fibrin sealant containing rEIG showed early wound constriction, lesser degree of inflammatory cells influx, and complete reepithelialization in 4-5 days, whereas the wound of control mice with the fibrin sealant alone showed contrary delayed reepithelialization, greater degree of inflammatory cell infiltration, and consequencial formation of greater granulation tissue at wound site. Taken together, these data suggest paradoxical effects of rEIG on the wound healing where in the wound exposed to infiltrating milieu of microorganisms in the oral cavity, the rEIG aggravates the wound healing by interfering with other innate defensive factors and extended greater flux of PMNs to inflamed wound site, while in the wound enclosed by fibrin, the rEIG accelerated wound healing by inhibiting the inflammation-generated proteases and the acute inflammatory reaction.     

A Rho Kinase (ROCK) Inhibitor,Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.     

EGCG Inhibits Adipose-Derived Mesenchymal Stem Cells Differentiation into Adipocytes and Prevents a STAT3-Mediated Paracrine Oncogenic Control of Triple-Negative Breast Cancer Cell Invasive Phenotype

Obese subjects have an increased risk of developing triple-negative breast cancer (TNBC), in part associated with the chronic low-grade inflammation state. On the other hand, epidemiological data indicates that increased consumption of polyphenol-rich fruits and vegetables plays a key role in reducing incidence of some cancer types. Here, we tested whether green tea-derived epigallocatechin-3-gallate (EGCG) could alter adipose-derived mesenchymal stem cell differentiation into adipocytes, and how this impacts the secretome profile and paracrine regulation of the TNBC invasive phenotype. Here, cell differentiation was performed and conditioned media (CM) from preadipocytes and mature adipocytes harvested. Human TNBC-derived MDA-MB-231 real-time cell migration was performed using the exCELLigence system. Differential gene arrays and RT-qPCR were used to assess gene expression levels. Western blotting was used to assess protein expression and phosphorylation status levels. In vitro vasculogenic mimicry (VM) was assessed with Matrigel. EGCG was found to inhibit the induction of key adipogenic biomarkers, including lipoprotein lipase, adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. Increased TNBC-derived MDA-MB-231 cell chemotaxis and vasculogenic mimicry were observed in response to mature adipocytes secretome, and this was correlated with increased STAT3 phosphorylation status. This invasive phenotype was prevented by EGCG, the JAK/STAT inhibitors Tofacitinib and AG490, as well as upon STAT3 gene silencing. In conclusion, dietary catechin-mediated interventions could, in part through the inhibition of adipogenesis and modulation of adipocytes secretome profile, prevent the onset of an obesogenic environment that favors TNBC development.     

Growth factor-expressing human neural progenitor cell grafts protect motor neurons but do not ameliorate motor performance and survival in ALS mice

Neural progenitor cells (NPs) have shown several promising benefits for the treatment of neurological disorders. To evaluate the therapeutic potential of human neural progenitor cells (hNPs) in amyotrophic lateral sclerosis (ALS), we transplanted hNPs or growth factor (GF)-expressing hNPs into the central nervous system (CNS) of mutant Cu/Zn superoxide dismutase (SOD1^G93A) transgenic mice. The hNPs were engineered to express brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), VEGF, neurotrophin-3 (NT-3), or glial cell-derived neurotrophic factor (GDNF), respectively, by adenoviral vector and GDNF by lentiviral vector before transplantation. Donor-derived cells engrafted and migrated into the spinal cord or brain of ALS mice and differentiated into neurons, oligodendrocytes, or glutamate transporter-1 (GLT1)-expressing astrocytes while some cells retained immature markers. Transplantation of GDNF- or IGF-1-expressing hNPs attenuated the loss of motor neurons and induced trophic changes in motor neurons of the spinal cord. However, improvement in motor performance and extension of lifespan were not observed in all hNP transplantation groups compared to vehicle-injected controls. Moreover, the lifespan of GDNF-expressing hNP recipient mice by lentiviral vector was shortened compared to controls, which was largely due to the decreased survival times of female animals. These results imply that although implanted hNPs differentiate into GLT1-expressing astrocytes and secrete GFs, which maintain dying motor neurons, inadequate trophic support could be harmful and there is sexual dimorphism in response to GDNF delivery in ALS mice. Therefore, additional therapeutic approaches may be required for full functional recovery.     

Analysis of mouse beta-haptoglobin chain by lectin affinoblotting detection

Two different human mammary carcinoma cell lines were xenotransplanted into nude mice. Serum samples were obtained prior to and after transplantation and investigated by two-dimensional electrophoresis (2-DE). By comparison of these silver-stained patterns additional protein spots were detected resulting either from proteins secreted or shed by the tumor itself or from mouse proteins induced by the tumor or the transplantation procedure. One group of spots detectable in post-transplantation serum as well as in control serum after mock-transplantation but not in pretransplantation serum was microsequenced and identified as mouse beta-haptoglobin. The carbohydrate structures of beta-haptoglobin were characterized by two different immunochemical glycoprotein staining procedures to detect differential terminal glycan modifications.     

Multi-Material Scaffolds for Tissue Engineering

A trend in developing biocompatible scaffolds for tissue engineering has been to seek an ideal single material for which a given cell type will exhibit favorable behavior. While an ideal single material has proven elusive, scaffold manufacture using combinations of specialist materials can produce more versatile structures. By controlling the percentage and architecture of material components, mechanical properties, cell attachment, and proliferation may be optimized for a given function. Three specialist materials, poly-ϵ-caprolactone (PCL), fibrin, and alginate, were incorporated into multi-component scaffolds for a series of experiments testing each component with culture of fibroblasts. The rigid and formable PCL provided structure, the fibrin pore-filler allowed for cell attachment, and alginate thread provided a nutrient transfer pathway in lieu of a vascular system. The efficacy of these scaffolds was judged on fibroblast distribution and population after 7-12 days of culture.     

Local Release of VEGF Using Fiber Mats Enables Effective Transplantation of Layered Cardiomyocyte Sheets

Cell sheet transplantation is a key tissue engineering technology. A vascular endothelial growth factor (VEGF)‐releasing fiber mat is developed for the transplantation of multilayered cardiomyocyte sheets. Poly(vinyl alcohol) fiber mats bearing poly(lactic‐co‐glycolic acid) nanoparticles that incorporate VEGF are fabricated using electrospinning and electrospray methods. Six‐layered cardiomyocyte sheets are transplanted with a VEGF‐releasing mat into athymic rats. After two weeks, these sheets produce thicker cardiomyocyte layers compared with controls lacking a VEGF‐releasing mat, and incorporate larger‐diameter blood vessels containing erythrocytes. Thus, local VEGF release near the transplanted cardiomyocytes induces vascularization, which supplies sufficient oxygen and nutrients to prevent necrosis. In contrast, cardiomyocyte sheets without a VEGF‐releasing mat do not survive in vivo, probably undergo necrosis, and are reduced in thickness. Hence, these VEGF‐releasing mats enable the transplantation of multilayered cardiomyocyte sheets in a single procedure, and should expand the potential of cell sheet transplantation for therapeutic applications.

     

Evaluation of PEGylated fibrin as a three-dimensional biodegradable scaffold for ovarian tissue engineering

The most challenging task of creating a bioengineered ovary to restore fertility in cancer patients is choosing an appropriate biomaterial to encapsulate isolated preantral follicles and ovarian cells. In this study, as a biocompatible and biodegradable biomaterial containing fibrin-like bioactivity and manageable physical properties, PEGylated fibrin aims to encapsulate isolated ovarian stromal cells as a first step of creating an engineered ovarian tissue. For this purpose, human ovarian stromal cells were isolated from frozen-thawed ovarian tissue and cultured in the PEGylated fibrin hydrogels (PEG:Fib), which were fabricated by combining two different molar ratios of PEG:Fib (10:1 and 5:1) and two thrombin concentrations. The samples were analyzed at days 0 and 5 of in vitro for cell density, proliferation (Ki67), and apoptosis (caspase-3). Moreover, LIVE/DEAD and PrestoBlue assays assessed cell viability and proliferation on days 1, 3, and 5. The effect of PEGylation on the biodegradation behavior of fibrin was evaluated by measuring the remaining mass ratio of non-modified fibrin, PEG:Fib 10:1, and PEG:Fib 5:1 hydrogels after 1, 2, 3, 5, 8, 11, and 15 days. The results showed that PEGylated fibrin hydrogels enhanced scaffold stability and supported cell viability and proliferation. In addition, PEG:Fib 5:1 T50 indicated a significantly higher cell density dynamic and non-significantly lower expression of caspase-3 on day 5. Besides, uniformity of cell distribution inside the hydrogel and a tendency to a high rate of Ki67-positive cells was observed in PEG:Fib 10:1 T50 hydrogels. In conclusion, this study reveals the positive effects of PEGylated fibrin hydrogels on isolated human ovarian stromal cells. Based on such promising findings, we believe that this matrix should be tested to encapsulate isolated human ovarian follicles.     

Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system

Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-α/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti- apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.     

Atrophy of brown adipocytes in the adult mouse causes transformation into white adipocyte-like cells

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.     

Compound A Increases Cell Infiltration in Target Organs of Acute Graft-versus-Host Disease (aGVHD) in a Mouse Model

Systemic steroids are used to treat acute graft-versus-host disease (aGVHD) caused by allogenic bone marrow transplantation (allo-BMT); however, their prolonged use results in complications. Hence, new agents for treating aGVHD are required. Recently, a new compound A (CpdA), with anti-inflammatory activity and reduced side effects compared to steroids, has been identified. Here, we aimed to determine whether CpdA can improve the outcome of aGVHD when administered after transplantation in a mouse model (C57BL/6 in B6D2F1). After conditioning with 9Gy total body irradiation, mice were infused with bone marrow (BM) cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. The animals were subsequently treated (3 days/week) with 7.5 mg/kg CpdA from day +15 to day +28; the controls received 0.9% NaCl. Thereafter, the incidence and severity of aGVHD in aGVHD target organs were analyzed. Survival and clinical scores did not differ significantly; however, CpdA-treated animals showed high cell infiltration in the target organs. In bulk mixed lymphocyte reactions, CpdA treatment reduced the cell proliferation and expression of inflammatory cytokines and chemokines compared to controls, whereas levels of TNF, IL-23, chemokines, and chemokine receptors increased. CpdA significantly reduced proliferation in vitro but increased T cell infiltration in target organs.     

Hepatocyte organoids and cell transplantation: What the future holds

Historically, primary hepatocytes have been difficult to expand or maintain in vitro. In this review, we will focus on recent advances in establishing hepatocyte organoids and their potential applications in regenerative medicine. First, we provide a background on the renewal of hepatocytes in the homeostatic as well as the injured liver. Next, we describe strategies for establishing primary hepatocyte organoids derived from either adult or fetal liver based on insights from signaling pathways regulating hepatocyte renewal in vivo. The characteristics of these organoids will be described herein. Notably, hepatocyte organoids can adopt either a proliferative or a metabolic state, depending on the culture conditions. Furthermore, the metabolic gene expression profile can be modulated based on the principles that govern liver zonation. Finally, we discuss the suitability of cell replacement therapy to treat different types of liver diseases and the current state of cell transplantation of in vitro-expanded hepatocytes in mouse models. In addition, we provide insights into how the regenerative microenvironment in the injured host liver may facilitate donor hepatocyte repopulation. In summary, transplantation of in vitro-expanded hepatocytes holds great potential for large-scale clinical application to treat liver diseases.Subject terms: Adult stem cells, Self-renewal