Astgik Petrosyan

Injection of Amniotic Fluid Stem Cells Delays Progression of Renal Fibrosis

Injection of amniotic fluid stem cells ameliorates the acute phase of acute tubular necrosis in animals by promoting proliferation of injured tubular cells and decreasing apoptosis, but whether these stem cells could be of benefit in CKD is unknown. Here, we used a mouse model of Alport syndrome, Col4a5(-/-) mice, to determine whether amniotic fluid stem cells could modify the course of progressive renal fibrosis. Intracardiac administration of amniotic fluid stem cells before the onset of proteinuria delayed interstitial fibrosis and progression of glomerular sclerosis, prolonged animal survival, and ameliorated the decline in kidney function. Treated animals exhibited decreased recruitment and activation of M1-type macrophages and a higher proportion of M2-type macrophages, which promote tissue remodeling. Amniotic fluid stem cells did not differentiate into podocyte-like cells and did not stimulate production of the collagen IVa5 needed for normal formation and function of the glomerular basement membrane. Instead, the mechanism of renal protection was probably the paracrine/endocrine modulation of both profibrotic cytokine expression and recruitment of macrophages to the interstitial space. Furthermore, injected mice retained a normal number of podocytes and had better integrity of the glomerular basement membrane compared with untreated Col4a5(-/-) mice. Inhibition of the renin-angiotensin system by amniotic fluid stem cells may contribute to these beneficial effects. In conclusion, treatment with amniotic fluid stem cells may be beneficial in kidney diseases characterized by progressive renal fibrosis.

Renal Extracellular Matrix Scaffolds From Discarded Kidneys Maintain Glomerular Morphometry and Vascular Resilience and Retains Critical Growth Factors.

Background Extracellular matrix (ECM) scaffolds, obtained through detergent-based decellularization of native kidneys, represent the most promising platform for investigations aiming at manufacturing kidneys for transplant purposes. We previously showed that decellularization of the human kidney yields renal ECM scaffolds (hrECMs) that maintain their basic molecular components, are cytocompatible, stimulate angiogenesis, and show an intact innate vasculature. However, evidence that the decellularization preserves glomerular morphometric characteristics, physiological parameters (pressures and resistances of the vasculature bed), and biological properties of the renal ECM, including retention of important growth factors (GFs), is still missing. Methods To address these issues, we studied the morphometry and resilience of hrECMs’ native vasculature with resin casting at electronic microscopy and pulse-wave measurements, respectively. Moreover, we determined the fate of 40 critical GFs post decellularization with a glass chip-based multiplex enzyme-linked immunosorbent assay array and in vitro immunofluorescence. Results Our method preserves the 3-dimensional conformation of the native glomerulus. Resin casting and pulse-wave measurements, showed that hrECMs preserves the microvascular morphology and morphometry, and physiological function. Moreover, GFs including vascular endothelial growth factor and its receptors are retained within the matrices. Conclusions Our results indicate that discarded human kidneys are a suitable source of renal scaffolds because they maintain a well-preserved structure and function of the vasculature, as well as GFs that are fundamental to achieve a satisfying recellularization of the scaffold in vivo due to their angiogenic properties.

A Novel Source of Cultured Podocytes

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.

A step towards clinical application of acellular matrix: A clue from macrophage polarization.

The outcome of tissue engineered organ transplants depends on the capacity of the biomaterial to promote a pro-healing response once implanted in vivo. Multiple studies, including ours, have demonstrated the possibility of using the extracellular matrix (ECM) of animal organs as platform for tissue engineering and more recently, discarded human organs have also been proposed as scaffold source. In contrast to artificial biomaterials, natural ECM has the advantage of undergoing continuous remodeling which allows adaptation to diverse conditions. It is known that natural matrices present diverse immune properties when compared to artificial biomaterials. However, how these properties compare between diseased and healthy ECM and artificial scaffolds has not yet been defined. To answer this question, we used decellularized renal ECM derived from WT mice and from mice affected by Alport Syndrome at different time-points of disease progression as a model of renal failure with extensive fibrosis. We characterized the morphology and composition of these ECMs and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid, PLLA). We showed that ECM derived from Alport kidneys differed in fibrous protein deposition and cytokine content when compared to ECM derived from WT kidneys. Yet, both WT and Alport renal ECM induced macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Anti-inflammatory properties of natural ECMs were lost when homogenized, hence three-dimensional structure of ECM seems crucial for generating an anti-inflammatory response. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as ideal scaffold for tissue engineering.

Direct Isolation and Characterization of Human Nephron Progenitors

Mature nephrons originate from a small population of uninduced nephrogenic progenitor cells (NPs) within the cap mesenchyme. These cells are characterized by the coexpression of SIX2 and CITED1. Many studies on mouse models as well as on human pluripotent stem cells have advanced our knowledge of NPs, but very little is known about this population in humans, since it is exhausted before birth and strategies for its direct isolation are still limited. Here we report an efficient protocol for direct isolation of human NPs without genetic manipulation or stepwise induction procedures. With the use of RNA‐labeling probes, we isolated SIX2+CITED1+ cells from human fetal kidney for the first time. We confirmed their nephrogenic state by gene profiling and evaluated their nephrogenic capabilities in giving rise to mature renal cells. We also evaluated the ability to culture these cells without complete loss of SIX2 and CITED1 expression over time. In addition to defining the gene profile of human NPs, this in vitro system facilitates studies of human renal development and provides a novel tool for renal regeneration and bioengineering purposes. Stem Cells Translational Medicine 2017;6:419–433

Amniotic fluid stem cell-derived vesicles protect from VEGF-induced endothelial damage

Injection of amniotic fluid stem cells (AFSC) delays the course of progression of renal fibrosis in animals with Alport Syndrome, enhancing kidney function and improving survival. The mechanisms responsible for these protective outcomes are still largely unknown. Here, we showed that vascular endothelial growth factor (VEGF) signaling within the glomeruli of Alport mice is strongly elevated early on in the disease, causing glomerular endothelial cell damage. Intraventricular injected AFSC that homed within the glomeruli showed strong modulation of the VEGF activity, particularly in glomerular endothelial cells. To investigate this phenomenon we hypothesized that extracellular vesicles (EVs) produced by the AFSC could be responsible for the observed renoprotection. AFSC derived EVs presented exosomal and stem cell markers on their surface membrane, including VEGFR1 and VEGFR2. EVs were able to modulate VEGF in glomerular endothelial cells by effectively trapping the excess VEGF through VEGFR1-binding preventing cellular damage. In contrast, VEGFR1/sVEGFR1 knockout EVs failed to show similar protection, thus indicating that VEGF trapping is a potentially viable mechanism for AFSC-EV mediated renoprotection. Taken together, our findings establish that EVs secreted by AFSC could target a specific signaling pathway within the glomerulus, thus representing a new potential glomerulus-specific targeted intervention.

PD46-06 NOVEL INSIGHTS ON WILM'S TUMOR: USING HUMAN NEPHRON PROGENITORS

RESULTS: Sorafenib/Sunitinib monotherapy, combined or alternating treatment groups demonstrated more significant anti-tumor activity, as compared to Axitinib monotherapy (P<0.05). Most interestingly, the PDX of KI2368, in contrast to KI2367, demonstrated significant anti-tumor activity to Sunitinib monotherapy, Sorafenib and Sunitinib combination and alternating treatment groups, but not to Sorafenib or Axitinib monotherapy (P<0.05). A total of 1725 genes have > 5-fold higher expression levels in KI2367 than in KI2368, including the relevant drug targets: PDGFA, PDGFB and PDGFRA. A total of 994 genes have > 5-fold higher expression in KI2368 than in KI2367. Against human reference genome, 5539 and 5827 protein change variants were respectively found in KI2367 and KI2368, 4023 common variants in both samples. We also found 20 and 4 in-frame gene fusions in KI2367 and KI2368, but no common in-frame fusion was detected. CONCLUSIONS: These results suggest the presence of the intra-tumor molecular heterogeneity in this patient, which could influence the clinical outcome of targeted therapies. Multiple biopsy and genomic analysis of intra-tumor molecular heterogeneity could potentially help guide more effective plan in selecting targeted therapies for ccRCC patients.

MP81-01 EFFECT OF INTEGRIN SIGNALING BLOCKADE ON SELF-RENEWAL AND DIFFERENTIATION OF HUMAN NEPHROGENIC PROGENITORS IN VITRO

INTRODUCTION AND OBJECTIVES: Mammalian kidney development is controlled through the proliferation and differentiation of a specific population of nephron progenitors (NP) characterized by expression of CITED1 and SIX2. The mechanisms regulating the balance between self-renewal and renal differentiation in NP are still elusive, impairing our ability to effectively expand NP in vitro for long term. In particular, the effect of extracellular matrix (ECM) composition and ECM-NP interaction are poorly understood. METHODS: We have investigated the relationship between ECM and self-renewal traits in NP isolated from human fetal kidneys (hFK). NP were isolated using our established RNA Smartflare protocol. Nephrogenic characteristics were confirmed by RNA-seq and nephrogenic potential by in vitro differentiation and dissociation/reaggregation assays. By immunofluorescence we have characterized the ECM present within the nephrogenic niche of hFK. Subsequently we tested NP expansion on these ECM substrates. RESULTS: Among others, laminin alpha 5, collagen 16 and collagen 18 where found to be highly expressed within the cap mesenchyme of developing hFK. In vitro, laminin was confirmed to better preserve self-renewal properties in NP, as confirmed by maintenance of higher co-expression of SIX2 and CITED1 in cultured NP. Interestingly, blocking integrin mediated ECM-NP interaction with specific antibodies lead to an increase in SIX2 and CITED1 co-expression, suggesting an important role of integrin mediated signaling pathway on balance between renal specification vs self-renewal. Effect of integrin blocking in NP on expression of SIX2 and CITED1 was further confirmed by PCR array, suggesting a direct role of ECM-NP interaction on self-renewal. CONCLUSIONS: Our data indicate a strong link between ECMNP during human renal development. NP-laminin interaction appears to play an essential role on nephron endowment by directly controlling self-renewal/differentiation balance. These results could provide not only a tool for the optimization of in vitro NP expansion but also a platform to advance our understanding of human renal development and nephron cell commitment.

Amniotic Fluid Cells: Kidney Injury and Regeneration

Summary The amniotic fluid (AF) is an invaluable source of organ-specific progenitor cells and stem cells that are easily obtainable, expandable over multiple passages, and can be used in various regenerative medicine applications. In the last few years, many studies have focused on the characterization of these cells, providing evidence of their capability of differentiating into various mature cell types, including those of the renal compartment. In addition, many papers have shown the in vivo application of these cells as modulators of disease progression both in acute and chronic damaged organs. Specifically, in this chapter we will focus our attention on the role of amniotic cells in renal regeneration. It has been shown that stem cells/progenitors from AF can contribute to functional recovery of both glomerular and tubular compartment and preliminary investigations have demonstrated their use for renal tissue engineering. Based on their peculiar characteristics these cells could represent a valuable tool for renal regenerative medicine applications, drug screening, and disease modeling studies.