Alveolar Type 2 Epithelial Cell Organoids: Focus on Culture Methods.

Lung diseases rank third in terms of mortality and represent a significant economic burden globally. Scientists have been conducting research to better understand respiratory diseases and find treatments for them. An ideal in vitro model must mimic the in vivo organ structure, physiology, and pathology. Organoids are self-organizing, three-dimensional (3D) structures originating from adult stem cells, embryonic lung bud progenitors, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). These 3D organoid cultures may provide a platform for exploring tissue development, the regulatory mechanisms related to the repair of lung epithelia, pathophysiological and immunomodulatory responses to different respiratory conditions, and screening compounds for new drugs. To create 3D lung organoids in vitro, both co-culture and feeder-free methods have been used. However, there exists substantial heterogeneity in the organoid culture methods, including the sources of AT2 cells, media composition, and feeder cell origins. This article highlights the currently available methods for growing AT2 organoids and prospective improvements to improve the available culture techniques/conditions. Further, we discuss various applications, particularly those aimed at modeling human distal lung diseases and cell therapy.

Role of soluble urokinase type plasminogen activator receptor (suPAR) in predicting mortality, readmission, length of stay and discharge in emergency patients: A systematic review and meta analysis.

BACKGROUND: Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker that is used to predict mortality, readmission, early discharge, and LOS, thus, serves as a useful tool for ED physicians. Our study aims to analyze the efficacy of suPAR in predicting these prognostic markers in ED. METHODS: We performed a comprehensive search on 6 databases from the inception to 30th November 2022, to select the following eligibility criteria; a) observation or triage trial studies investigating the role of suPAR levels in predicting: 30 day and 90-day mortality, 30-day readmission, early discharge (within 24hr), and LOS in patients coming to AMU. RESULTS: A total of 13 studies were included, with a population size of 35,178, of which 52.9% were female with a mean age of 62.93 years. Increased risk of 30-day mortality (RR = 10.52; 95% CI = 4.82-22.95; I2 = 38%; P < .00001), and risk of 90-day mortality (RR = 5.76; 95% CI = 3.35-9.91; I2 = 36%; P < .00001) was observed in high suPAR patients. However, a slightly increased risk was observed for 30-day readmission (RR = 1.50; 95% CI = 1.16-1.94; I2 = 54%; P = .002). More people were discharged within 24hr in the low suPAR level group compared to high suPAR group (RR = 0.46; 95% CI = 0.40-0.53; I2 = 41%; P < .00001). LOS was thrice as long in high suPAR level patients than in patients with low suPAR (WMD = 3.20; 95% CI = 1.84-4.56; I2 = 99%; P < .00001). CONCLUSION: suPAR is proven to be a significant marker in predicting 30-day and 90-day mortality in ED patients.

PAI-1 regulates AT2-mediated re-alveolarization and ion permeability.

BACKGROUND: Acute lung injury is characterized by overwhelmingly elevated PAI-1 in both lung edema fluid and the circulating system. The role of increased PAI-1, encoded by Serpine1 gene, in the regeneration of injured lung epithelium has not been understood completely. This study aimed to investigate the role of Serpine1 in the regulation of alveolar type 2 epithelial cell (AT2) fate in a humanized mouse line carrying diseased mutants (Serpine1Tg). METHODS: Wild-type (wt) and Serpine1Tg AT2 cells were either cultured as monolayers or 3D alveolospheres. Colony-forming assay and total surface area of organoids were analyzed. AT1 and AT2 cells in organoids were counted by immunohistochemistry and fluorescence-activated cell sorting (FACS). To test the potential effects of elevated PAI-1 on the permeability in the epithelial monolayers, we digitized the biophysical properties of polarized AT2 monolayers grown at the air-liquid interface. RESULTS: A significant reduction in total AT2 cells harvested in Serpine1Tg mice was observed compared with wt controls. AT2 cells harvested from Serpine1Tg mice reduced significantly over the wt controls. Spheroids formed by Serpine1Tg AT2 cells were lesser than wt control. Similarly, the corresponding surface area, a readout of re-alveolarization of injured epithelium, was markedly reduced in Serpine1Tg organoids. FACS analysis revealed a significant suppression in the number of AT2 cells, in particular, the CD44+ subpopulation, in Serpine1Tg organoids. A lesser ratio of AT1:AT2 cells in Serpine1Tg organoids was observed compared with wt cultures. There was a significant increase in transepithelial resistance but not amiloride inhibition. CONCLUSIONS: Our study suggests elevated PAI-1 in injured lungs downregulates alveolar epithelial regeneration by reducing the AT2 self-renewal, particularly in the CD44+ cells.

An unusual foreign body ricocheting into the pericardium after a penetrating thoracic injury: A case report.

INTRODUCTION AND IMPORTANCE: Injuries to the thorax are common in trauma patients, among whom cardiac injuries are most lethal, particularly in the setting of penetrating trauma. CASE PRESENTATION: In this case report, the foreign body was visualized to be lodged in between the left atrium and ventricle. After that, an emergency open heart surgery was performed. Post-intervention, the patient was shifted to the intensive care unit without ionotropic support and with stable haemodynamics. CLINICAL DISCUSSION: Cardiac foreign bodies are rarely seen because most patients with penetrating cardiac injuries die from hemorrhagic shock or cardiac tamponade. CONCLUSION: We report a case of a penetrating injury into the thorax with the foreign body being lodged into the pericardium and then managed surgically. The patient was discharged subsequently and followed up after a few days with progressive recovery.

PAI-1 regulates AT2-mediated re-alveolarization and ion permeability.

Background Acute lung injury is characterized by overwhelmingly elevated PAI-1 in both lung edema fluid and the circulating system. The role of increased PAI-1, encoded by Serpine1 gene, in the regeneration of injured lung epithelium has not been understood completely. This study aimed to investigate the role of Serpine1 in the regulation of alveolar type 2 epithelial cell (AT2) fate in a humanized mouse line carrying diseased mutants (Serpine1Tg). Methods Wild type (wt) and Serpine1Tg AT2 cells were either cultured as monolayers or 3D alveolospheres. Colony forming assay and total surface area of organoids were analyzed. AT1 and AT2 cells in organoids were counted by immunohistochemistry and fluorescence-activated cell sorting (FACS). To test the potential effects of elevated PAI-1 on the permeability in the epithelial monolayers, we digitized the biophysical properties of polarized AT2 monolayers grown at the air-liquid interface. Results A significant reduction in total AT2 cells harvested in Serpine1Tg mice was observed compared with wt controls. AT2 cells harvested from Serpine1Tg mice reduced significantly over the wt controls. Spheroids formed by Serpine1Tg AT2 cells were lesser than wt control. Similarly, the corresponding surface area, a readout of realveolarization of injured epithelium, was markedly reduced in Serpine1Tg organoids. FACS analysis revealed a significant suppression in the number of AT2 cells, in particular, the CD44+ subpopulation, in Serpine1Tg organoids. A lesser ratio of AT1:AT2 cells in Serpine1Tg organoids was observed compared with wt cultures. There was a significant increase in transepithelial resistance but not amiloride inhibition. Conclusions Our study suggests elevated PAI-1 in injured lungs downregulates alveolar epithelial regeneration by reducing the AT2 self-renewal, particularly in the CD44+ cells.

3D tissue-engineered lung models to study immune responses following viral infections of the small airways.

Small airway infections caused by respiratory viruses are some of the most prevalent causes of illness and death. With the recent worldwide pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is currently a push in developing models to better understand respiratory diseases. Recent advancements have made it possible to create three-dimensional (3D) tissue-engineered models of different organs. The 3D environment is crucial to study physiological, pathophysiological, and immunomodulatory responses against different respiratory conditions. A 3D human tissue-engineered lung model that exhibits a normal immunological response against infectious agents could elucidate viral and host determinants. To create 3D small airway lung models in vitro, resident epithelial cells at the air-liquid interface are co-cultured with fibroblasts, myeloid cells, and endothelial cells. The air-liquid interface is a key culture condition to develop and differentiate airway epithelial cells in vitro. Primary human epithelial and myeloid cells are considered the best 3D model for studying viral immune responses including migration, differentiation, and the release of cytokines. Future studies may focus on utilizing bioreactors to scale up the production of 3D human tissue-engineered lung models. This review outlines the use of various cell types, scaffolds, and culture conditions for creating 3D human tissue-engineered lung models. Further, several models used to study immune responses against respiratory viruses, such as the respiratory syncytial virus, are analyzed, showing how the microenvironment aids in understanding immune responses elicited after viral infections.

Prkg2 regulates alveolar type 2-mediated re-alveolarization.

BACKGROUND: The cGMP-dependent type 2 protein kinase, encoded by the prkg2 gene, is highly expressed in alveolar type 2 epithelial (AT2) cells. It is unclear whether prkg2 regulates AT2 cell homeostasis and re-alveolarization of injured lungs. This study aimed to investigate the role of prkg2 in the regulation of the fate of AT2 in vitro. METHODS: Primary AT2 cells of wild-type (wt) and prkg2-/- mice were co-cultured with fibroblasts as three-dimensional organoids. The colony formation was analyzed between days 4 and 12 post-seeding. EdU assay was used to detect cells with active DNA synthesis. AT1 and AT2 cells in organoids were visualized with anti-podoplanin and anti-surfactant protein C antibodies, respectively. RESULTS: Prkg2-/- AT2 cells developed a greater number of organoids than wt controls. However, compared to wt organoids, a lower number of AT2 but a greater number of AT1 cells were visualized. In addition, a lower number of proliferated cells (EdU+) were observed in prkg2-/- organoids compared to wt controls. The numbers of organoids and EdU+ cells were significantly reduced in protein kinase A (PKA) inhibitor H89-treated wt and prkg2-/- cultures. Organoids and EdU+ cells were increased by lipopolysaccharides (LPS) in both wt and prkg2-/- groups. The increase in the proportion of AT1 and AT2 cells in organoids was only seen in wt controls. CONCLUSIONS: Prkg2 may regulate the lineage of AT2 cells, which is affected by endotoxins and the interactive PKA signaling pathway.

Quantifying the effect of roadway, driver, vehicle, and location characteristics on the frequency of longitudinal and lateral accelerations.

The purpose of this study is to understand and quantify the simultaneous effects of roadway speed category, driver age, driver gender, vehicle class, and location on the rates of longitudinal and lateral acceleration epochs. The rate of usual as well as harsh acceleration epochs are used to extract insights on driving risk and driver comfort preferences. However, an analysis of acceleration rates at multiple thresholds incorporating various effects while using a large-scale and diverse dataset is missing. This analysis will fill this research gap. Data from the 2nd Strategic Highway Research Program Naturalistic Driving Study (SHRP2 NDS) was used for this analysis. The rate of occurrence of acceleration epochs was modeled using negative binomial distribution based generalized linear mixed effect models. Roadway speed category, driver age, driver gender, vehicle class, and location were used as the fixed effects and the driver identifier was used as the random effect. Incidence rate ratios were then calculated to compare subcategories of each fixed effect. Roadway speed category has the strongest effect on longitudinal and lateral accelerations of all magnitudes. Acceleration epoch rates consistently decrease as the roadway speed category increases. The difference in the rates depends on the threshold and is up to three orders of magnitude. Driver age is another significant factor with clear trends for longitudinal and lateral acceleration epochs. Younger and older drivers experience higher rates of longitudinal accelerations and decelerations. However, the rate of lateral accelerations consistently decreases with age. Vehicle class also has a significant effect on the rate of harsh accelerations with minivans consistently experiencing lower rates.

Advances in genome editing: the technology of choice for precise and efficient ß-thalassemia treatment.

Beta (ß)-thalassemia is one of the most significant hemoglobinopathy worldwide. The high prevalence of the ß-thalassemia carriers aggravates the disease burden for patients and national economies in the developing world. The survival of ß-thalassemia patients solely relies on repeated transfusions, which eventually results into multi-organ damage. The fetal γ-globin genes are ordinarily silenced at birth and replaced by the adult ß-globin genes. However, mutations that cause lifelong persistence of fetal γ-globin, ameliorate the debilitating effects of ß-globin mutations. Therefore, therapeutically reactivating the fetal γ-globin gene is a prime focus of researchers. CRISPR/Cas9 is the most common approach to correct disease causative mutations or to enhance or disrupt the expression of proteins to mitigate the effects of the disease. CRISPR/cas9 and prime gene editing to correct mutations in hematopoietic stem cells of ß-thalassemia patients has been considered a novel therapeutic approach for effective hemoglobin production. However, genome-editing technologies, along with all advantages, have shown some disadvantages due to either random insertions or deletions at the target site of edition or non-specific targeting in genome. Therefore, the focus of this review is to compare pros and cons of these editing technologies and to elaborate the retrospective scope of gene therapy for ß-thalassemia patients.

Immunosuppressant-Induced Oxidative Stress and Iron: A Paradigm Shift from Systemic to Intrahepatic Abnormalities.

Immunosuppressants are used clinically to lower rejection rates in transplant patients. Unfortunately, the adverse side effects of these immunosuppressants can be severe, which is one of the rationales that life expectancy of individuals after transplant still significantly falls short of that of the general population. The current experimental setup was designed to analyze the tacrolimus-induced hepatic iron overload in Wistar rats. Four experimental groups were orally given 1 ml of aqueous suspension of tacrolimus (12 mg/kg) through oral gavage, and rats were sacrificed after 6, 12, 24, and 48 h of tacrolimus dose. Hepatic hepcidin expression was found to be significantly augmented along with the upregulation of Tf and TfR1, Ferritin-L, Ferritin-H, TNF-α, and HO-1 gene expression at 6 and 12 h, and downregulation of Fpn-1, Hjv, and Heph at 6 h was detected. Significant downregulation of IL-6, IFN-α, IFN-ß, and IFN-γ at all study time points was also observed. Serum iron level was decreased while serum hepcidin level was found to be significantly increased. Iron staining showed blue-stained hemosiderin granules within the hepatocytes, sinusoidal spaces, and portal areas at 12 and 24 h time points and remarkable fall of iron contents in the splenic red pulp. These results suggest that the use of tacrolimus leads to the onset of an intrahepatic acute-phase response-like reaction and causes iron overload in hepatic cells by altering the expression of key proteins involved in iron metabolism.

Plasmin improves blood-gas barrier function in oedematous lungs by cleaving epithelial sodium channels.

BACKGROUND AND PURPOSE: Lung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. Here, we have tested whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid. EXPERIMENTAL APPROACH: Human lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, and protein docking were combined for identifying plasmin cleavage sites. KEY RESULTS: Plasmin improved lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin activated αßγENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts (αΔ432-444, γΔ131-138, γΔ178-193, and γΔ410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human γENaC proteins. In addition, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both α and γ subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal. CONCLUSION AND IMPLICATIONS: Plasmin can cleave ENaC to improve blood-gas exchange by resolving oedema fluid and could be a potent therapy for oedematous lungs.

Elevated Neurokinin-1 Receptor Expression in Uterine Products of Conception Is Associated With First Trimester Miscarriages.

BACKGROUND: Miscarriage is a common complication of early pregnancy, mostly occurring in the first trimester. However, the etiological factors and prognostic and diagnostic biomarkers are not well known. Neurokinin-1 receptor (NK-1R) is a receptor of tachykinin peptide substance P (SP) and has a role in various pathological conditions, cancers, but its association with miscarriages and significance as a clinicopathological parameter are not studied. Accordingly, the present study aimed to clarify the localization and expression for NK-1R in human retained products of conception (POC). The role of NK-1R is not known in miscarriages. MATERIALS AND METHODS: NK-1R expression was assessed in POC and normal placental tissues by immunohistochemistry. Three- to four-micrometer-thin sections of formalin-fixed paraffin-embedded tissues were used for this purpose. Tissues were processed and then immunohistochemically stained with NK-1R antibody. Brain tissue was used as control for antibody. Protein expression was evaluated using the nuclear labeling index (%). Tissues were counterstained with 3,3'-diaminobenzidine (DAB), and microscopy was performed at 10×, 20×, and 40× magnifications. RESULTS: Ten human POC tissues and 10 normal placental tissues were studied by immunohistochemistry to demonstrate the localization of NK-1R. The expression of NK-1R protein was high in all the cases of both groups. NK-1R expression showed no notable differences among different cases of miscarriages as well as normal deliveries at full term regardless of the mother's age and gestational age at which the event occurred. Statistically, no difference was found in both groups, which is in agreement with our hypothesis and previous findings. CONCLUSION: The expression of NK-1R was similar in all the cases, and it was intense. It shows that dysregulation of NK-1R along with its ligand SP might be involved in miscarriages and also involved in normal delivery. Our results provide fundamental data regarding this anti-NK-1R strategy. Thus, the present study recommends that SP/NK-1R system might, therefore, be considered as an emerging and promising diagnostic and therapeutic strategy against miscarriages. Hence, we report for the first time the expression and localization of NK-1R in POC. We suggest NK-1R antagonist in addition to the immunoglobulins and human chorionic gonadotropin to diagnose and treat spontaneous miscarriages.

Proliferative regulation of alveolar epithelial type 2 progenitor cells by human Scnn1d gene.

Lung epithelial sodium channel (ENaC) encoded by Scnn1 genes is essential for maintaining transepithelial salt and fluid homeostasis in the airway and the lung. Compared to α, ß, and γ subunits, the role of respiratory δ-ENaC has not been studied in vivo due to the lack of animal models. Methods: We characterized full-length human δ802-ENaC expressed in both Xenopus oocytes and humanized transgenic mice. AT2 proliferation and differentiation in 3D organoids were analysed with FACS and a confocal microscope. Both two-electrode voltage clamp and Ussing chamber systems were applied to digitize δ802-ENaC channel activity. Immunoblotting was utilized to analyse δ802-ENaC protein. Transcripts of individual ENaC subunits in human lung tissues were quantitated with qPCR. Results: The results indicate that δ802-ENaC functions as an amiloride-inhibitable Na+ channel. Inhibitory peptide α-13 distinguishes δ802- from α-type ENaC channels. Modified proteolysis of γ-ENaC by plasmin and aprotinin did not alter the inhibition of amiloride and α-13 peptide. Expression of δ802-ENaC at the apical membrane of respiratory epithelium was detected with biophysical features similar to those of heterologously expressed channels in oocytes. δ802-ENaC regulated alveologenesis through facilitating the proliferation of alveolar type 2 epithelial cells. Conclusion: The humanized mouse line conditionally expressing human δ802-ENaC is a novel model for studying the expression and function of this protein in vivo .

Hepatoprotective effect of ketoconazole in chronic liver injury model.

Ketoconazole is a first orally available anti-fungal drug which has been reported as a potent inhibitor of human cytochrome P-450. The present study was designed to examine the heptoprotective effect of ketoconazole in both in vitro and in vivo liver injury models. Hepatocyte injury was induced by 8mM CCl4 while hepatic fibrosis model was established by injecting 1 ml/kg CCl4 followed by treatment with ketoconazole. Effect of ketoconazole treatment on injured hepatocytes was determined by lactate dehydrogenase release and trypan blue assay. Analysis of ketoconazole treatment and prevention on liver fibrosis was assessed by sirius red staining, masson trichome staining, PCR and liver function tests for bilirubin and alanine transaminase (ALAT).A significant reduction (P<0.05) in LDH release and reduced number of dead cells was observed in hepatocytes treated with ketoconazole. Sirus red and masson trichome stainings showed reduced levels of collagen in both treated and preventive groups and down regulation of alpha smooth muscle actin was observed with up-regulations of MMP-2, CK-8 and CK-18. Hepatic functional assessment demonstrated reduced serum levels of bilirubin and ALAT. Treatment of fibrotic liver with ketoconazole improves hepatic microenvironment and enhanced reduction of liver injury after fibrosis. Cytochrome P-450 inhibitors seems a favored therapeutic option in attenuation of liver fibrosis.

Fagonia indica Repairs Hepatic Damage through Expression Regulation of Toll-Like Receptors in a Liver Injury Model.

Fagonia indica is a traditionally used phytomedicine to cure hepatic ailments. However, efficient validation of its hepatoprotective effect and molecular mechanisms involved are not yet well established. Therefore, the present study was designed to evaluate the hepatoprotective activity of Fagonia indica and to understand the molecular mechanisms involved in the reversal of hepatic injury. The liver injury mouse model was established by thioacetamide followed by oral administration of plant extract. Serum biochemical and histological analyses were performed to assess the level of hepatic injury. Expression analysis of proinflammatory, hepatic, and immune regulatory genes was performed with RT-PCR. Results of serological and histological analyses described the restoration of normal liver function and architecture in mice treated with plant extract. In addition, altered expression of proinflammatory (IL-1ß, IL-6, TNF-α, and TGF-ß) and hepatic (krt-18 and albumin) markers further strengthens the liver injury reversal effects of Fagonia indica. Furthermore, a significant expression regulation of innate immunity components such as toll-like receptors 4 and 9 and MyD-88 was observed suggesting an immune regulatory role of the plant in curing liver injury. In conclusion, the current study not only proposes Fagonia indica, a strong hepatoprotective candidate, but also recommends an immune regulatory toll-like receptor pathway as an important therapeutic target in liver diseases.

Serum from CCl4-induced acute rat injury model induces differentiation of ADSCs towards hepatic cells and reduces liver fibrosis.

Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl4 injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl4 which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl4-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson's trichome staining was observed.

Expression profiling of adhesion proteins during prenatal and postnatal liver development in rats.

Culturing of primary hepatocytes and stem cell-derived hepatocytes faces a major issue of dedifferentiation due to absence of cell-cell adhesion and 3D structures. One of the possible ways to eliminate the problem of dedifferentiation is mimicking the expression pattern of adhesion proteins during the normal developmental process of liver cells. The purpose of this study was to evaluate the expression pattern of some key adhesion proteins, namely, E-cadherin, N-cadherin, epithelial CAM (EpCAM), intracellular CAM (ICAM), collagen 1α1, α-actinin, ß-catenin and vimentin, in the liver tissue during prenatal and postnatal stages. Furthermore, differences in their expression between prenatal, early postnatal and adult stages were highlighted. Wistar rats were used to isolate livers at prenatal Day 14 and 17 as well as on postnatal Day 1, 3, 7 and 14. The liver from adult rats was used as control. Both conventional and real-time quantitative polymerase chain reactions (PCRs) were performed. For most of the adhesion proteins such as E-cadherin, N-cadherin, EpCAM, ICAM, collagen 1α1 and α-actinin, low expression was observed around prenatal Day 14 and an increasing expression was observed in the postnatal period. Moreover, ß-catenin and vimentin showed higher expression in the early prenatal period, which decreased gradually in the postnatal period, but still this low expression was considerably higher than that in the adult control rats. This basic knowledge of the regulation of expression of adhesion proteins during different developmental stages indicates their vital role in liver development. This pattern can be further studied and imitated under in vitro conditions to achieve better cell-cell interactions.

Differentiation of Mesenchymal Stem Cells Towards Nephrogenic Lineage and Their Enhanced Resistance to Oxygen Peroxide-induced Oxidative Stress.

INTRODUCTION: Mesenchymal stem cells (MSCs) have been publicized to ameliorate kidney injury both in vitro and in vivo. However, very less is known if MSCs can be differentiated towards renal lineages and their further application potential in kidney injuries. MATERIALS AND METHODS: The present study developed a conditioning system of growth factors fibroblast growth factor 2, transforming growth factor-ß2, and leukemia inhibitory factor for in vitro differentiation of MSCs isolated from different sources towards nephrogenic lineage. Less invasively isolated adipose-derived MSCs were also compared to bone marrow-derived MSCs for their differentiation potential to induce renal cell. Differentiated MSCs were further evaluated for their resistance to oxidative stress induced by oxygen peroxide. RESULTS: A combination of growth factors successfully induced differentiation of MSCs. Both types of differentiated cells showed significant expression of pronephrogenic markers (Wnt4, Wt1, and Pax2) and renal epithelial markers (Ecad and ZO1). In contrast, expression of mesenchymal stem cells marker Oct4 and Vim were downregulated. Furthermore, differentiated adipose-derived MSCs and bone marrow-derived MSCs showed enhanced and comparable resistance to oxygen peroxide-induced oxidative stress. CONCLUSIONS: Adipose-derived MSC provides a promising alternative to bone marrow-derived MSC as a source of autologous stem cells in human kidney injuries. In addition, differentiated MSCs with further in vivo investigations may serve as a cell source for tissue engineering or cell therapy in different renal ailments.

In vitro differentiated hepatic oval-like cells enhance hepatic regeneration in CCl4 -induced hepatic injury.

Hepatic oval cells are likely to be activated during advanced stage of liver fibrosis to reconstruct damaged hepatic tissue. However, their scarcity, difficulties in isolation, and in vitro expansion hampered their transplantation in fibrotic liver. This study was aimed to investigate the repair potential of in vitro differentiated hepatic oval-like cells in CCl4 -induced liver fibrosis. BMSCs and oval cells were isolated and characterized from C57BL/6 GFP+ mice. BMSCs were differentiated into oval cells by preconditioning with HGF, EGF, SCF, and LIF and analyzed for the oval cells-specific genes. Efficiency of oval cells to reduce hepatocyte injury was studied by determining cell viability, release of LDH, and biochemical tests in a co-culture system. Further, in vivo repair potential of differentiated oval cells was determined in CCl4 -induced fibrotic model by gene expression analysis, biochemical tests, mason trichrome, and Sirius red staining. Differentiated oval cells expressed hepatic oval cells-specific markers AFP, ALB, CK8, CK18, CK19. These differentiated cells when co-cultured with injured hepatocytes showed significant hepato-protection as measured by reduction in apoptosis, LDH release, and improvement in liver functions. Transplantation of differentiated oval cells like cells in fibrotic livers exhibited enhanced homing, reduced liver fibrosis, and improved liver functions by augmenting hepatic microenvironment by improved liver functions. This preconditioning strategy to differentiate BMSCs into oval cell leads to improved survival and homing of transplanted cells. In addition, reduction in fibrosis and functional improvement in mice with CCl4 -induced liver fibrosis was achieved.