Identification and Characterization of the Wilms Tumor Cancer Stem Cell.

A nephrogenic progenitor cell (NP) with cancer stem cell characteristics driving Wilms tumor (WT) using spatial transcriptomics, bulk and single cell RNA sequencing, and complementary in vitro and transplantation experiments is identified and characterized. NP from WT samples with NP from the developing human kidney is compared. Cells expressing SIX2 and CITED1 fulfill cancer stem cell criteria by reliably recapitulating WT in transplantation studies. It is shown that self-renewal versus differentiation in SIX2+CITED1+ cells is regulated by the interplay between integrins ITGß1 and ITGß4. The spatial transcriptomic analysis defines gene expression maps of SIX2+CITED1+ cells in WT samples and identifies the interactive gene networks involved in WT development. These studies define SIX2+CITED1+ cells as the nephrogenic-like cancer stem cells of WT and points to the renal developmental transcriptome changes as a possible driver in regulating WT formation and progression.

Regenerative medicine applications: An overview of clinical trials.

Insights into the use of cellular therapeutics, extracellular vesicles (EVs), and tissue engineering strategies for regenerative medicine applications are continually emerging with a focus on personalized, patient-specific treatments. Multiple pre-clinical and clinical trials have demonstrated the strong potential of cellular therapies, such as stem cells, immune cells, and EVs, to modulate inflammatory immune responses and promote neoangiogenic regeneration in diseased organs, damaged grafts, and inflammatory diseases, including COVID-19. Over 5,000 registered clinical trials on ClinicalTrials.gov involve stem cell therapies across various organs such as lung, kidney, heart, and liver, among other applications. A vast majority of stem cell clinical trials have been focused on these therapies' safety and effectiveness. Advances in our understanding of stem cell heterogeneity, dosage specificity, and ex vivo manipulation of stem cell activity have shed light on the potential benefits of cellular therapies and supported expansion into clinical indications such as optimizing organ preservation before transplantation. Standardization of manufacturing protocols of tissue-engineered grafts is a critical first step towards the ultimate goal of whole organ engineering. Although various challenges and uncertainties are present in applying cellular and tissue engineering therapies, these fields' prospect remains promising for customized patient-specific treatments. Here we will review novel regenerative medicine applications involving cellular therapies, EVs, and tissue-engineered constructs currently investigated in the clinic to mitigate diseases and possible use of cellular therapeutics for solid organ transplantation. We will discuss how these strategies may help advance the therapeutic potential of regenerative and transplant medicine.

Persistent COUP-TFII expression underlies the myopathy and impaired muscle regeneration observed in resistance to thyroid hormone-alpha.

Thyroid hormone signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury, via activation of thyroid nuclear receptor alpha (THRA). A mouse model of resistance to thyroid hormone carrying a frame-shift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury. The expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA-PV mice and it is known to negatively regulate myogenesis. Here, we report that in murine myoblasts COUP-TFII interacts with THRA and modulates THRA binding to thyroid response elements (TREs). Silencing of COUP-TFII expression restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts. Moreover, COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice.

Circulating B Cells With Memory and Antibody-Secreting Phenotypes Are Detectable in Pediatric Kidney Transplant Recipients Before the Development of Antibody-Mediated Rejection.

Development of anti-human leukocyte antigen donor-specific antibodies (DSAs) is associated with antibody-mediated rejection (AMR) and reduced allograft survival in kidney transplant recipients. Whether changes in circulating lymphocytes anticipate DSA or AMR development is unclear. METHODS: We used time-of-flight mass cytometry to analyze prospectively collected peripheral blood mononuclear cells (PBMC) from pediatric kidney transplant recipients who developed DSA (DSA-positive recipients [DSAPOS], n = 10). PBMC were obtained at 2 months posttransplant, 3 months before DSA development, and at DSA detection. PBMC collected at the same time points posttransplant from recipients who did not develop DSA (DSA-negative recipients [DSANEG], n = 11) were used as controls. RESULTS: DSAPOS and DSANEG recipients had similar baseline characteristics and comparable frequencies of total B and T cells. Within DSAPOS recipients, there was no difference in DSA levels (mean fluorescence intensity [MFI]: 13 687 ± 4159 vs 11 375 ± 1894 in DSAPOSAMR-positive recipients (AMRPOS) vs DSAPOSAMR-negative recipients (AMRNEG), respectively; P = 0.630), C1q binding (5 DSAPOSAMRPOS [100%] vs 4 DSAPOSAMRNEG [80%]; P = 1.000), or C3d binding (3 DSAPOSAMRPOS [60%] vs 1 DSAPOSAMRNEG [20%]; P = 0.520) between patients who developed AMR and those who did not. However, DSAPOS patients who developed AMR (n = 5; 18.0 ± 3.6 mo post-DSA detection) had increased B cells with antibody-secreting (IgD-CD27+CD38+; P = 0.002) and memory (IgD-CD27+CD38-; P = 0.003) phenotypes compared with DSANEG and DSAPOSAMRNEG recipients at DSA detection. CONCLUSIONS: Despite the small sample size, our comprehensive phenotypic analyses show that circulating B cells with memory and antibody-secreting phenotypes are present at DSA onset, >1 year before biopsy-proven AMR in pediatric kidney transplant recipients.

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.

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.

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.