Kidney regeneration and repair after transplantation

PURPOSE OF REVIEW: To briefly show which are the mechanisms and cell types involved in kidney regeneration and describe some of the therapies currently under study in regenerative medicine for kidney transplantation. RECENT FINDINGS: The kidney contains cell progenitors that under specific circumstances have the ability to regenerate specific structures. Apart from the knowledge gained in the self-regenerative properties of the kidney, new concepts in regenerative medicine such as organ engineering and the use of mesenchymal stem cell-based therapies are currently the focus of attention in the field. SUMMARY: Overall, kidney regeneration is a reality and the knowledge on how to control it will be one of the main scopes in the present and future

Toward development of imesenchymal stem cells for immunomodulatory therapy

Mesenchymal stem cells (MSC) are under development as an immunomodulatory therapy. The anticipated immunomodulatory effects of MSC are broad, from direct inhibition of lymphocyte proliferation, induction of regulatory T and B cells, to resetting the immune system via a hit-and-run principle. There are endless flavors of MSC. Differences between MSC are originating from donors variation, differences in tissue of origin, the effects of culture conditions, and expansion time. Even standard culture conditions change the properties of MSC dramatically and generate MSC that only remotely resemble their in vivo counterparts. Adjustments in culture protocols can further emphasize properties of interest in MSC, thereby generating cells fitted for specific purposes. Culture improved immunomodulatory MSC can be designed to target particular immune disorders. In this review, we describe the observed and the desired immunomodulatory effects of MSC and propose approaches how MSC with optimal immunomodulatory properties can be developed

Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV.The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 mu m). As a control, non-conditioned culture medium was used (control medium).To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV

Inflammatory conditions dictate the effect of mesenchymal stem or stromal cells on B cell function

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is, however, unknown how MSC interact with B cells under inflammatory conditions. In this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43- selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40, and IL-2; and after 7 days, B cell proliferation, phenotype, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38high CD24high B cells (Bregs) and IL-10 production, while MSC-IFN-γ significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions, MSC stimulate Breg induction; whereas, under inflammatory conditions, MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pretreatment of MSC

Mesenchymal Stem Cell Therapy Prevents Interstitial Fibrosis and Tubular Atrophy in a Rat Kidney Allograft Model

In solid organ transplantation, mesenchymal stem cell (MSC) therapy is strongly emerging among other cell therapies due to the positive results obtained in vitro and in vivo as an immunomodulatory agent and their potential regenerative role. We aimed at testing whether a single dose of MSCs, injected at 11 weeks after kidney transplantation for the prevention of chronic mechanisms, enhanced regeneration and provided protection against the inflammatory and fibrotic processes that finally lead to the characteristic features of chronic allograft nephropathy (CAN). Either bone marrow mononuclear cells (BMCs) injection or no-therapy (NT) were used as control treatments. A rat kidney transplantation model of CAN with 2.5 h of cold ischemia was used, and functional, histological, and molecular parameters were assessed at 12 and 24 weeks after transplantation. MSC and BMC cell therapy preserves renal function at 24 weeks and abrogates proteinuria, which is typical of this model (NT24w: 68.9 +/- 26.5mg/24 h, MSC24w: 16.6 +/- 2.3mg/24 h, BMC24w: 24.1 +/- 5.3mg/24 h, P < 0.03). Only MSC-treated animals showed a reduction in interstitial fibrosis and tubular atrophy (NT24w: 2.3 +/- 0.29, MSC24w: 0.4 +/- 0.2, P < 0.03), less T cells (NT: 39.6 +/- 9.5, MSC: 8.1 +/- 0.9, P < 0.03) and macrophages (NT: 20.9 +/- 4.7, MSC: 5.9 +/- 1.7, P < 0.05) infiltrating the parenchyma and lowered expression of inflammatory cytokines while increasing the expression of anti-inflammatory factors. MSCs appear to serve as a protection from injury development rather than regenerate the damaged tissue, as no differences were observed in Ki67 expression, and kidney injury molecule-1, Clusterin, NGAL, and hepatocyte growth factor expression were only up-regulated in nontreated animals. Considering the results, a single delayed MSC injection is effective for the long-term protection of kidney allografts

N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV-Cell Interaction and Functional Activation of Endothelial Cells

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV-cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells' interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton's Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV-cell interactions and associated functions

Different Storing and Processing Conditions of Human Lymphocytes do not Alter P-Glycoprotein Rhodamine 123 Efflux

P-glycoprotein (Pgp), a protein codified by Multi Drug Resistance (MDR1) gene, has a detoxifying function and might influence the toxicity and pharmacokinetics and pharmacodynamics of drugs. Sampling strategies to improve Pgp studies could be useful to optimize the sensitivity and the reproducibility of efflux assays. This study aimed to compare Pgp expression and efflux activity by measuring Rhodamine123 (Rh123) retention in lymphocytes stored under different conditions, in order to evaluate the potential utility of any of the storing conditions in Pgp functionality. Our results show no change in protein expression of Pgp by confocal studies and Western blotting, nor changes at the mRNA level (qRT-PCR). No differences in Rh123 efflux by Pgp activity assays were found between fresh and frozen lymphocytes after 24 hours of blood extraction, using either of the two Pgp specific inhibitors (VP and PSC833). Different working conditions in the 24 hours post blood extraction do not affect Rh123 efflux. These results allow standardization of Pgp activity measurement in different individuals with different timing of blood sampling and in different geographic areas. ______________

Pediatric mesenchymal stem cells exhibit immunomodulatory properties toward allogeneic T and B cells under inflammatory conditions

Mesenchymal stem cells from pediatric patients (pMSCs) are an attractive cell source in regenerative medicine, due to their higher proliferation rates and better differentiation abilities compared to adult MSCs (aMSCs). We have previously characterized the immunomodulatory abilities of pMSCs on T cells under co-culture. It has also been reported that aMSCs can inhibit B cell proliferation and maturation under inflammatory conditions. In this study, we therefore aimed to clarify the immunomodulatory effect of pMSCs toward T and B cells in an inflammatory microenvironment. Bone marrow derived pMSCs were primed to simulate inflammatory conditions by exposure with 50 ng/mL of IFN-γ for 3 days. To analyze the interaction between pMSCs and T cells, CD3/CD28 stimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with primed or unprimed pMSCs. To investigate B cell responses, quiescent B cells obtained from spleens by CD43 negative selection were stimulated with anti-IgM, anti-CD40, IL-2, and co-cultured with either IFN-γ primed or unprimed pMSC. pMSC phenotype, B and T cell proliferation, and B cell functionality were analyzed. Gene expression of indoleamine 2,3-dioxygenease (IDO), as well as the expression of HLA-ABC, HLA-DR and the co-stimulatory molecules CD80 and CD86 was upregulated on pMSCs upon IFN-γ priming. IFN-γ did not alter the immunomodulatory abilities of pMSCs upon CD4+ nor CD8+ stimulated T cells compared to unprimed pMSCs. IFN-γ primed pMSCs but not unprimed pMSCs strongly inhibited naïve (CD19+CD27-), memory (CD19+CD27+), and total B cell proliferation. Antibody-producing plasmablast (CD19+CD27highCD38high) formation and IgG production were also significantly inhibited by IFN-γ primed pMSCs compared to unprimed pMSCs. Collectively, these results show that pMSCs have immunomodulatory effects upon the adaptive immune response which can be potentiated by inflammatory stimuli. This knowledge is useful in regenerative medicine and allogeneic transplantation applications toward tailoring pMSCs function to best modulate the immune response for a successful implant engraftment and avoidance of a strong immune reaction