Pannexin-3 stabilizes the transcription factor Bcl6 in a channel-independent manner to protect against vascular oxidative stress.

Targeted degradation regulates the activity of the transcriptional repressor Bcl6 and its ability to suppress oxidative stress and inflammation. Here, we report that abundance of endothelial Bcl6 is determined by its interaction with Golgi-localized pannexin 3 (Panx3) and that Bcl6 transcriptional activity protects against vascular oxidative stress. Consistent with data from obese, hypertensive humans, mice with an endothelial cell-specific deficiency in Panx3 had spontaneous systemic hypertension without obvious changes in channel function, as assessed by Ca2+ handling, ATP amounts, or Golgi luminal pH. Panx3 bound to Bcl6, and its absence reduced Bcl6 protein abundance, suggesting that the interaction with Panx3 stabilized Bcl6 by preventing its degradation. Panx3 deficiency was associated with increased expression of the gene encoding the H2O2-producing enzyme Nox4, which is normally repressed by Bcl6, resulting in H2O2-induced oxidative damage in the vasculature. Catalase rescued impaired vasodilation in mice lacking endothelial Panx3. Administration of a newly developed peptide to inhibit the Panx3-Bcl6 interaction recapitulated the increase in Nox4 expression and in blood pressure seen in mice with endothelial Panx3 deficiency. Panx3-Bcl6-Nox4 dysregulation occurred in obesity-related hypertension, but not when hypertension was induced in the absence of obesity. Our findings provide insight into a channel-independent role of Panx3 wherein its interaction with Bcl6 determines vascular oxidative state, particularly under the adverse conditions of obesity.

Targeting Bcl6 in the TREX1 D18N murine model ameliorates autoimmunity by modulating T-follicular helper cells and germinal center B cells.

The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post-apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus-like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus-like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T-follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage-defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B-cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1-associated or other lupus-like diseases.

Advances and Challenges in Kidney Organoids.

The advent of organoids has renewed researchers' interest in in vitro cell culture systems. A wide variety of protocols, primarily utilizing pluripotent stem cells, are under development to improve organoid generation to mimic organ development. The complexity of organoids generated is greatly influenced based on the method used. Understanding the process of kidney organoid formation gives developmental insights into how renal cells form, mature, and interact with the adjacent cells to form specific spatiotemporal structural patterns. This knowledge can bridge the gaps in understanding in vivo renal developmental processes. Evaluating genetic and epigenetic signatures in specialized cell types can help interpret the molecular mechanisms governing cell fate. In addition, development in single-cell RNA sequencing, 3D bioprinting and microfluidic technologies has led to better identification and understanding of a variety of cell types during differentiation and designing of complex structures to mimic the conditions in vivo. While several reviews have highlighted the application of kidney organoids, there is no comprehensive review of various methodologies specifically focusing on kidney organoids. This review summarizes the updated differentiation methodologies, applications, and challenges associated with kidney organoids. Here we have comprehensively collated all the different variables influencing the organoid generation.

Hybrid cytokine IL233 renders protection in murine acute graft vs host disease (aGVHD).

Previously, we generated IL233, a hybrid cytokine composed of interleukin (IL)-2 and IL-33, with better therapeutic potential than either cytokine in multiple inflammatory diseases, in part through promoting T-regulatory cells (Tregs). Here we test the potential of IL233 pretreatment in a murine model of excessive Th1 activation, the parent-into-F1 model of acute GVHD (aGVHD). Five days of IL233 pretreatment of the recipients blocked or delayed the aGVHD-linked loss of B cells as seen in either the peripheral blood (day-11) or lymph nodes (day-14). IL233 pretreatment also prevented the expansion of donor CD8 T-cells in blood and LN at day-14 and significantly reduced day-14 serum IFNγ and TNFα compared to saline treated GVHD mice although, the level of Tregs did not statistically differ between saline and IL233-treated mice. Overall, the current study provides support for the use of IL233 as a therapeutic option in excessive Th1/CD8-driven conditions.

Targeting Regulatory T Cells for Therapy of Lupus Nephritis.

Lupus glomerulonephritis (LN) is a complex autoimmune disease characterized by circulating autoantibodies, immune-complex deposition, immune dysregulation and defects in regulatory T cell (Tregs). Treatment options rely on general immunosuppressants and steroids that have serious side effects. Approaches to target immune cells, such as B cells in particular, has had limited success and new approaches are being investigated. Defects in Tregs in the setting of autoimmunity is well known and Treg-replacement strategies are currently being explored. The aim of this minireview is to rekindle interest on Treg-targeting strategies. We discuss the existing evidences for Treg-enhancement strategies using key cytokines interleukin (IL)-2, IL-33 and IL-6 that have shown to provide remission in LN. We also discuss strategies for indirect Treg-modulation for protection from LN.

The Yin and Yang of Alarmins in Regulation of Acute Kidney Injury.

Acute kidney injury (AKI) is a major clinical burden affecting 20 to 50% of hospitalized and intensive care patients. Irrespective of the initiating factors, the immune system plays a major role in amplifying the disease pathogenesis with certain immune cells contributing to renal damage, whereas others offer protection and facilitate recovery. Alarmins are small molecules and proteins that include granulysins, high-mobility group box 1 protein, interleukin (IL)-1α, IL-16, IL-33, heat shock proteins, the Ca++ binding S100 proteins, adenosine triphosphate, and uric acid. Alarmins are mostly intracellular molecules, and their release to the extracellular milieu signals cellular stress or damage, generally leading to the recruitment of the cells of the immune system. Early studies indicated a pro-inflammatory role for the alarmins by contributing to immune-system dysregulation and worsening of AKI. However, recent developments demonstrate anti-inflammatory mechanisms of certain alarmins or alarmin-sensing receptors, which may participate in the prevention, resolution, and repair of AKI. This dual function of alarmins is intriguing and has confounded the role of alarmins in AKI. In this study, we review the contribution of various alarmins to the pathogenesis of AKI in experimental and clinical studies. We also analyze the approaches for the therapeutic utilization of alarmins for AKI.

Novel Immunomodulatory Cytokine Regulates Inflammation, Diabetes, and Obesity to Protect From Diabetic Nephropathy.

Obesity-linked (type 2) diabetic nephropathy (T2DN) has become the largest contributor to morbidity and mortality in the modern world. Recent evidences suggest that inflammation may contribute to the pathogenesis of T2DN and T-regulatory cells (Treg) are protective. We developed a novel cytokine (named IL233) bearing IL-2 and IL-33 activities in a single molecule and demonstrated that IL233 promotes Treg and T-helper (Th) 2 immune responses to protect mice from inflammatory acute kidney injury. Here, we investigated whether through a similar enhancement of Treg and inhibition of inflammation, IL233 protects from T2DN in a genetically obese mouse model, when administered either early or late after the onset of diabetes. In the older mice with obesity and microalbuminuria, IL233 treatment reduced hyperglycemia, plasma glycated proteins, and albuminuria. Interestingly, IL233 administered before the onset of microalbuminuria not only strongly inhibited the progression of T2DN and reversed diabetes as indicated by lowering of blood glucose, normalization of glucose tolerance and insulin levels in islets, but surprisingly, also attenuated weight gain and adipogenicity despite comparable food intake. Histological examination of kidneys showed that saline control mice had severe inflammation, glomerular hypertrophy, and mesangial expansion, which were all attenuated in the IL233 treated mice. The protection correlated with greater accumulation of Tregs, group 2 innate lymphoid cells (ILC2), alternately activated macrophages and eosinophils in the adipose tissue, along with a skewing toward T-helper 2 responses. Thus, the novel IL233 cytokine bears therapeutic potential as it protects genetically obese mice from T2DN by regulating multiple contributors to pathogenesis. Short Description: A novel bifunctional cytokine IL233, bearing IL-2 and IL-33 activities reverses inflammation and protects from type-2 diabetic nephropathy through promoting T-regulatory cells and type 2 immune response.

IL233, an IL-2-IL-33 hybrid cytokine induces prolonged remission of mouse lupus nephritis by targeting Treg cells as a single therapeutic agent.

Lupus glomerulonephritis (GN) is an autoimmune disease characterized by immune complex-deposition, complement activation and glomerular inflammation. In lupus-prone NZM2328 mice, the occurrence of lupus GN was accompanied by a decrease in Treg cells and an increase in proinflammatory cytokine-producing T cells. Because IL-33 in addition to IL-2 has been shown to be important for Treg cell proliferation and ST2 (IL-33 receptor) positive Treg cells are more potent in suppressor activity, a hybrid cytokine with active domains of IL-2 and IL-33 was generated to target the ST2+ Treg cells as a therapeutic agent to treat lupus GN. Three mouse models were used: spontaneous and Ad-IFNα- accelerated lupus GN in NZM2328 and the lymphoproliferative autoimmune GN in MRL/lpr mice. Daily injections of IL233 for 5 days prevented Ad-IFNα-induced lupus GN and induced remission of spontaneous lupus GN. The remission was permanent in that no relapses were detected. The remission was accompanied by persistent elevation of Treg cells in the renal lymph nodes. IL233 is more potent than IL-2 and IL-33 either singly or in combination in the treatment of lupus GN. The results of this study support the thesis that IL233 should be considered as a novel agent for treating lupus GN.

A Novel Hybrid Cytokine IL233 Mediates regeneration following Doxorubicin-Induced Nephrotoxic Injury.

Kidney injury, whether due to ischemic insults or chemotherapeutic agents, is exacerbated by inflammation, whereas Tregs are protective. We recently showed that IL-2 and IL-33, especially as a hybrid cytokine (IL233 - bearing IL-2 and IL-33 activities in one molecule), potentiated Tregs and group 2 innate lymphoid cells (ILC2) to prevent renal injury. Recent studies have indicated a reparative function for Tregs and ILC2. Here, using doxorubicin-induced nephrotoxic renal injury model, we investigated whether IL233 administration either before, late or very late after renal injury can restore kidney structure and function. We found that IL233 treatment even 2-weeks post-doxorubicin completely restored kidney function accompanied with an increase Treg and ILC2 in lymphoid and renal compartments, augmented anti-inflammatory cytokines and attenuated proinflammatory cytokine levels. IL233 treated mice had reduced inflammation, kidney injury (Score values - saline: 3.34 ± 0.334; IL233 pre: 0.42 ± 0.162; IL233 24 hrs: 1.34 ± 0.43; IL233 1 week: 1.2 ± 0.41; IL233 2 week: 0.47 ± 0.37; IL233 24 hrs + PC61: 3.5 ± 0.74) and fibrosis in all treatment regimen as compared to saline controls. Importantly, mice treated with IL233 displayed a reparative program in the kidneys, as evidenced by increased expression of genes for renal progenitor-cells and nephron segments. Our findings present the first evidence of an immunoregulatory cytokine, IL233, which could be a potent therapeutic strategy that augments Treg and ILC2 to not only inhibit renal injury, but also promote regeneration.

IL233, A Novel IL-2 and IL-33 Hybrid Cytokine, Ameliorates Renal Injury.

CD4+Foxp3+ regulatory T cells (Tregs) protect the kidney during AKI. We previously found that IL-2, which is critical for Treg homeostasis, upregulates the IL-33 receptor (ST2) on CD4+ T cells, thus we hypothesized that IL-2 and IL-33 cooperate to enhance Treg function. We found that a major subset of Tregs in mice express ST2, and coinjection of IL-2 and IL-33 increased the number of Tregs in lymphoid organs and protected mice from ischemia-reperfusion injury (IRI) more efficiently than either cytokine alone. Accordingly, we generated a novel hybrid cytokine (IL233) bearing the activities of IL-2 and IL-33 for efficient targeting to Tregs. IL233 treatment increased the number of Tregs in blood and spleen and prevented IRI more efficiently than a mixture of IL-2 and IL-33. Injection of IL233 also increased the numbers of Tregs in renal compartments. Moreover, IL233-treated mice had fewer splenic Tregs and more Tregs in kidneys after IRI. In vitro, splenic Tregs from IL233-treated mice suppressed CD4+ T cell proliferation better than Tregs from saline-treated controls. IL233 treatment also improved the ability of isolated Tregs to inhibit IRI in adoptive transfer experiments and protected mice from cisplatin- and doxorubicin-induced nephrotoxic injury. Finally, treatment with IL233 increased the proportion of ST2-bearing innate lymphoid cells (ILC2) in blood and kidneys, and adoptive transfer of ILC2 also protected mice from IRI. Thus, the novel IL233 hybrid cytokine, which utilizes the cooperation of IL-2 and IL-33 to enhance Treg- and ILC2-mediated protection from AKI, bears strong therapeutic potential.

Therapeutic Application of Placental Mesenchymal Stem Cells Reprogrammed Neurospheres in Spinal Cord Injury of SCID.

Mesenchymal stromal cells (MSCs) and induced pluripotent stem cells (iPSCs) have stimulated much interest in the scientific community and hopes among the general public since their discovery in 1966 due to a variety of potential applications it has in the field of regenerative medicine. Copious amount of literature, as well as long-term animal and human clinical trials, indicates that MSCs can be successfully used for therapeutic purpose without any extreme adversities. MSCs have been isolated from adult and fetal tissues. Recently, MSCs from placenta have generated much inquisitiveness. In this article, we will demonstrate the step-by-step procedure for isolating human placental MSCs from term placenta, reprogramming of placental MSCs into iPSCs using plasmid vectors, evaluation of functional recovery in mice spinal cord injury models, and in vivo tracking of the transplanted cells.

Therapeutic Application of Human Wharton Jelly Mesenchymal Stem Cells in Skin Injury of SCID.

Mesenchymal stem cells (MSCs) are blossoming as a credible source for regenerative medical applications. The use of fetal MSCs is gaining momentum for therapeutic use. The ease of isolation, enhanced characteristics, and immunomodulation properties renders the utilization of fetal MSCs for numerous clinical applications. In this article, we will demonstrate a step-by-step protocol for isolation of Wharton's jelly MSCs (WJMSCs) from the human umbilical cord matrix, preparation of human platelet lysate, fabricating amniotic membrane scaffold and mice model to study skin regeneration using a combination of MSCs and decellularized amniotic membrane scaffold.

hiPSC-derived iMSCs: NextGen MSCs as an advanced therapeutically active cell resource for regenerative medicine.

Mesenchymal stem cells (MSCs) are being assessed for ameliorating the severity of graft-versus-host disease, autoimmune conditions, musculoskeletal injuries and cardiovascular diseases. While most of these clinical therapeutic applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor remains limited. The utility of MSCs derived from human-induced pluripotent stem cells (hiPSCs) has been shown in recent pre-clinical studies. Since adult MSCs have limited capability regarding proliferation, the quantum of bioactive factor secretion and immunomodulation ability may be constrained. Hence, the alternate source of MSCs is being considered to replace the commonly used adult tissue-derived MSCs. The MSCs have been obtained from various adult and foetal tissues. The hiPSC-derived MSCs (iMSCs) are transpiring as an attractive source of MSCs because during reprogramming process, cells undergo rejuvination, exhibiting better cellular vitality such as survival, proliferation and differentiations potentials. The autologous iMSCs could be considered as an inexhaustible source of MSCs that could be used to meet the unmet clinical needs. Human-induced PSC-derived MSCs are reported to be superior when compared to the adult MSCs regarding cell proliferation, immunomodulation, cytokines profiles, microenvironment modulating exosomes and bioactive paracrine factors secretion. Strategies such as derivation and propagation of iMSCs in chemically defined culture conditions and use of footprint-free safer reprogramming strategies have contributed towards the development of clinically relevant cell types. In this review, the role of iPSC-derived mesenchymal stromal cells (iMSCs) as an alternate source of therapeutically active MSCs has been described. Additionally, we also describe the role of iMSCs in regenerative medical applications, the necessary strategies, and the regulatory policies that have to be enforced to render iMSC's effectiveness in translational medicine.

Quest for alternate personalized clinical source of MSCs: Advancing towards hiPSCs derived iMSCs.

The Human mesenchymal stromal/stem cells (MSCs) have been isolated from various tissue sources. Yet, the lack of a distinctive marker for identifying in vivo MSCs in their tissue niche has hampered the MSC's in vivo behavior tracking and compared that to the in vitro expanded cultures. In this review, we present a comprehensive report on MSCs history, isolation from assorted tissue sources, classification, long-term cultures for comprehensively characterized MSCs, immunomodulation, regenerative medical applications, iMSCs as a novel source of patient-specific iPSCs and scaleup strategies for translational applications. We have emphasized on prenatal tissue-derived MSCs and iMSCs derived from hiPSCs as an effective alternative to adult MSCs. We also highlight the urgent requirement to revisit the initial criteria laid down by International Society for Cellular Therapy (ISCT) and propose more stringent criteria to define, identify and exclusively characterize the MSCs derived from various tissue sources using advanced molecular tools; also more international workshops are necessary for delineating unique features of MSCs. Unless the proposed goal is achieved, it is extremely difficult to realize the full potential of MSCs in translational applications. Although numerous patients have been tested with MSCs to date, no immediate adverse outcomes or infusion-related toxicity has been reported, suggesting MSCs infusion to be safe. However, rare adverse event and late complications of the treatment may be detected in large cohorts of patients with long-term follow-up.

Cell Therapy Augments Functional Recovery Subsequent to Spinal Cord Injury under Experimental Conditions.

The spinal cord injury leads to enervation of normal tissue homeostasis ultimately leading to paralysis. Until now there is no proper cure for the treatment of spinal cord injury. Recently, cell therapy in animal spinal cord injury models has shown some progress of recovery. At present, clinical trials are under progress to evaluate the efficacy of cell transplantation for the treatment of spinal cord injury. Different types of cells such as pluripotent stem cells derived neural cells, mesenchymal stromal cells, neural stem cells, glial cells are being tested in various spinal cord injury models. In this review we highlight both the advances and lacuna in the field of spinal cord injury by discussing epidemiology, pathophysiology, molecular mechanism, and various cell therapy strategies employed in preclinical and clinical injury models and finally we discuss the limitations and ethical issues involved in cell therapy approach for treating spinal cord injury.

Noninvasive Optical Imaging and In Vivo Cell Tracking of Indocyanine Green Labeled Human Stem Cells Transplanted at Superficial or In-Depth Tissue of SCID Mice.

Stem cell based therapies hold great promise for the treatment of human diseases; however results from several recent clinical studies have not shown a level of efficacy required for their use as a first-line therapy, because more often in these studies fate of the transplanted cells is unknown. Thus monitoring the real-time fate of in vivo transplanted cells is essential to validate the full potential of stem cells based therapy. Recent studies have shown how real-time in vivo molecular imaging has helped in identifying hurdles towards clinical translation and designing potential strategies that may contribute to successful transplantation of stem cells and improved outcomes. At present, there are no cost effective and efficient labeling techniques for tracking the cells under in vivo conditions. Indocyanine green (ICG) is a safer, economical, and superior labelling technique for in vivo optical imaging. ICG is a FDA-approved agent and decades of usage have clearly established the effectiveness of ICG for human clinical applications. In this study, we have optimized the ICG labelling conditions that is optimal for noninvasive optical imaging and demonstrated that ICG labelled cells can be successfully used for in vivo cell tracking applications in SCID mice injury models.

Human Wharton's Jelly Mesenchymal Stem Cells plasticity augments scar-free skin wound healing with hair growth.

Human mesenchymal stem cells (MSCs) are a promising candidate for cell-based transplantation and regenerative medicine therapies. Thus in the present study Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) have been derived from extra embryonic umbilical cord matrix following removal of both arteries and vein. Also, to overcome the clinical limitations posed by fetal bovine serum (FBS) supplementation because of xenogeneic origin of FBS, usual FBS cell culture supplement has been replaced with human platelet lysate (HPL). Apart from general characteristic features of bone marrow-derived MSCs, wharton jelly-derived MSCs have the ability to maintain phenotypic attributes, cell growth kinetics, cell cycle pattern, in vitro multilineage differentiation plasticity, apoptotic pattern, normal karyotype-like intrinsic mesenchymal stem cell properties in long-term in vitro cultures. Moreover, the WJ-MSCs exhibited the in vitro multilineage differentiation capacity by giving rise to differentiated cells of not only mesodermal lineage but also to the cells of ectodermal and endodermal lineage. Also, WJ-MSC did not present any aberrant cell state upon in vivo transplantation in SCID mice and in vitro soft agar assays. The immunomodulatory potential assessed by gene expression levels of immunomodulatory factors upon exposure to inflammatory cytokines in the fetal WJ-MSCs was relatively higher compared to adult bone marrow-derived MSCs. WJ-MSCs seeded on decellularized amniotic membrane scaffold transplantation on the skin injury of SCID mice model demonstrates that combination of WJ-MSCs and decellularized amniotic membrane scaffold exhibited significantly better wound-healing capabilities, having reduced scar formation with hair growth and improved biomechanical properties of regenerated skin compared to WJ-MSCs alone. Further, our experimental data indicate that indocyanin green (ICG) at optimal concentration can be resourcefully used for labeling of stem cells and in vivo tracking by near infrared fluorescence non-invasive live cell imaging of labelled transplanted cells, thus proving its utility for therapeutic applications.

Long-term cultured human term placenta-derived mesenchymal stem cells of maternal origin displays plasticity.

Mesenchymal stem cells (MSCs) are an alluring therapeutic resource because of their plasticity, immunoregulatory capacity and ease of availability. Human BM-derived MSCs have limited proliferative capability, consequently, it is challenging to use in tissue engineering and regenerative medicine applications. Hence, placental MSCs of maternal origin, which is one of richest sources of MSCs were chosen to establish long-term culture from the cotyledons of full-term human placenta. Flow analysis established bonafied MSCs phenotypic characteristics, staining positively for CD29, CD73, CD90, CD105 and negatively for CD14, CD34, CD45 markers. Pluripotency of the cultured MSCs was assessed by in vitro differentiation towards not only intralineage cells like adipocytes, osteocytes, chondrocytes, and myotubules cells but also translineage differentiated towards pancreatic progenitor cells, neural cells, and retinal cells displaying plasticity. These cells did not significantly alter cell cycle or apoptosis pattern while maintaining the normal karyotype; they also have limited expression of MHC-II antigens and are Naive for stimulatory factors CD80 and CD 86. Further soft agar assays revealed that placental MSCs do not have the ability to form invasive colonies. Taking together all these characteristics into consideration, it indicates that placental MSCs could serve as good candidates for development and progress of stem-cell based therapeutics.