Generation of Induced Nephron Progenitor-like Cells from Human Urine-Derived Cells.

BACKGROUND: Regenerative medicine strategies employing nephron progenitor cells (NPCs) are a viable approach that is worthy of substantial consideration as a promising cell source for kidney diseases. However, the generation of induced nephron progenitor-like cells (iNPCs) from human somatic cells remains a major challenge. Here, we describe a novel method for generating NPCs from human urine-derived cells (UCs) that can undergo long-term expansion in a serum-free condition. RESULTS: Here, we generated iNPCs from human urine-derived cells by forced expression of the transcription factors OCT4, SOX2, KLF4, c-MYC, and SLUG, followed by exposure to a cocktail of defined small molecules. These iNPCs resembled human embryonic stem cell-derived NPCs in terms of their morphology, biological characteristics, differentiation potential, and global gene expression and underwent a long-term expansion in serum-free conditions. CONCLUSION: This study demonstrates that human iNPCs can be readily generated and expanded, which will facilitate their broad applicability in a rapid, efficient, and patient-specific manner, particularly holding the potential as a transplantable cell source for patients with kidney disease.

Long-term expansion of directly reprogrammed keratinocyte-like cells and in vitro reconstitution of human skin.

BACKGROUND: Human keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting. METHODS: Induced keratinocyte-like cells (iKCs) were directly reprogrammed from human urine cells by retroviral transduction of two lineage-specific transcription factors BMI1 and △NP63α (BN). Expression of keratinocyte stem cell or their differentiation markers were assessed by PCR, immunofluorescence and RNA-Sequencing. Regeneration capacity of iKCs were assessed by reconstitution of a human skin equivalent under air-interface condition. RESULTS: BN-driven iKCs were similar to primary keratinocytes (pKCs) in terms of their morphology, protein expression, differentiation potential, and global gene expression. Moreover, BN-iKCs self-assembled to form stratified skin equivalents in vitro. CONCLUSIONS: This study demonstrated an approach to generate human iKCs that could be directly reprogrammed from human somatic cells and extensively expanded in serum- and feeder cell-free systems, which will facilitate their broad applicability in an efficient and patient-specific manner.

Reprogramming human urine cells into intestinal organoids with long-term expansion ability and barrier function.

Generation of intestinal organoids from human somatic cells by reprogramming would enable intestinal regeneration, disease modeling, and drug screening in a personalized pattern. Here, we report a direct reprogramming protocol for the generation of human urine cells induced intestinal organoids (U-iIOs) under a defined medium. U-iIOs expressed multiple intestinal specific genes and showed resembling gene expression profiles to primary small intestines. U-iIOs can be stably long-term expanded and further differentiated into more mature intestinal lineage cells with high expression of metallothionein and cytochrome P450 (CYP450) genes. These specific molecular features of U-iIOs differ from human pluripotent stem cells derived intestinal organoids (P-iIOs) and intestinal immortalized cell lines. Furthermore, U-iIOs exhibit intestinal barriers indicated by blocking FITC-dextran permeation and uptaking of the specific substrate rhodamine 123. Our study provides a novel platform for patient-specific intestinal organoid generation, which may lead to precision treatment of intestinal diseases and facilitate drug discovery.

Urine-Derived Renal Epithelial Cells (URECs) from Transplanted Kidneys as a Promising Immunomodulatory Cell Population.

Kidney transplantation is a lifesaving procedure for patients with end-stage kidney disease (ESKD). Organs derived from donation after cardiac death (DCD) are constantly increasing; however, DCD often leads to ischaemia-reperfusion (IR) and Acute Kidney Injury (AKI) events. These phenomena increase kidney cell turnover to replace damaged cells, which are voided in urine. Urine-derived renal epithelial cells (URECs) are rarely present in the urine of healthy subjects, and their loss has been associated with several kidney disorders. The present study aimed to characterize the phenotype and potential applications of URECs voided after transplant. The results indicate that URECs are highly proliferating cells, expressing several kidney markers, including markers of kidney epithelial progenitor cells. Since the regulation of the immune response is crucial in organ transplantation and new immunoregulatory strategies are needed, UREC immunomodulatory properties were investigated. Co-culture with peripheral blood mononuclear cells (PBMCs) revealed that URECs reduced PBMC apoptosis, inhibited lymphocyte proliferation, increased T regulatory (Treg) cells and reduced T helper 1 (Th1) cells. URECs from transplanted patients represent a promising cell source for the investigation of regenerative processes occurring in kidneys, and for cell-therapy applications based on the regulation of the immune response.

MYOCD is Required for Cardiomyocyte-like Cells Induction from Human Urine Cells and Fibroblasts Through Remodeling Chromatin.

Despite direct reprogramming of human cardiac fibroblasts into induced cardiomyocytes (iCM) holds great potential for heart regeneration, the mechanisms are poorly understood. Whether other human somatic cells could be reprogrammed into cardiomyocytes is also unknown. Here, we report human urine cells (hUCs) could be converted into CM-like cells from different donors and the related chromatin accessibility dynamics (CAD) by assay for transposase accessible chromatin(ATAC)-seq. hUCs transduced by MEF2C, TBX5, MESP1 and MYOCD but without GATA4 expressed multiple cardiac specific genes, exhibited Ca2+ oscillation potential and sarcomeric structures, and contracted synchronously in coculture with mouse CM. Additionally, we found that MYOCD is required for both closing and opening critical loci, mainly by hindering the opening of loci enriched with motifs for the TEAD and AP1 family and promoting the closing of loci enriched with ETS motifs. These changes differ partially from CAD observed during iCM induction from human fibroblasts. Collectively, our study offers one practical platform for iCM generation and insights into mechanisms for iCM fate determination.

The Nephrotoxin Puromycin Aminonucleoside Induces Injury in Kidney Organoids Differentiated from Induced Pluripotent Stem Cells.

Kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), which can progress to end stage renal disease (ESRD), are a worldwide health burden. Organ transplantation or kidney dialysis are the only effective available therapeutic tools. Therefore, in vitro models of kidney diseases and the development of prospective therapeutic options are urgently needed. Within the kidney, the glomeruli are involved in blood filtration and waste excretion and are easily affected by changing cellular conditions. Puromycin aminonucleoside (PAN) is a nephrotoxin, which can be employed to induce acute glomerular damage and to model glomerular disease. For this reason, we generated kidney organoids from three iPSC lines and treated these with PAN in order to induce kidney injury. Morphological observations revealed the disruption of glomerular and tubular structures within the kidney organoids upon PAN treatment, which were confirmed by transcriptome analyses. Subsequent analyses revealed an upregulation of immune response as well as inflammatory and cell-death-related processes. We conclude that the treatment of iPSC-derived kidney organoids with PAN induces kidney injury mediated by an intertwined network of inflammation, cytoskeletal re-arrangement, DNA damage, apoptosis and cell death. Furthermore, urine-stem-cell-derived kidney organoids can be used to model kidney-associated diseases and drug discovery.

Generation of mitochondria-rich kidney organoids from expandable intermediate mesoderm progenitors reprogrammed from human urine cells under defined medium.

BACKGROUND: The kidneys require vast amounts of mitochondria to provide ample energy to reabsorb nutrients and regulate electrolyte, fluid, and blood pressure homeostasis. The lack of the human model hinders the investigation of mitochondria homeostasis related to kidney physiology and disease. RESULTS: Here, we report the generation of mitochondria-rich kidney organoids via partial reprogramming of human urine cells (hUCs) under the defined medium. First, we reprogrammed mitochondria-rich hUCs into expandable intermediate mesoderm progenitor like cells (U-iIMPLCs), which in turn generated nephron progenitors and formed kidney organoids in both 2D and 3D cultures. Cell fate transitions were confirmed at each stage by marker expressions at the RNA and protein levels, along with chromatin accessibility dynamics. Single cell RNA-seq revealed hUCs-induced kidney organoids (U-iKOs) consist of podocytes, tubules, and mesenchyme cells with 2D dominated with mesenchyme and 3D with tubule and enriched specific mitochondria function associated genes. Specific cell types, such as podocytes and proximal tubules, loop of Henle, and distal tubules, were readily identified. Consistent with these cell types, 3D organoids exhibited the functional and structural features of the kidney, as indicated by dextran uptake and transmission electron microscopy. These organoids can be further matured in the chick chorioallantoic membrane. Finally, cisplatin, gentamicin, and forskolin treatment led to anatomical abnormalities typical of kidney injury and altered mitochondria homeostasis respectively. CONCLUSIONS: Our study demonstrates that U-iKOs recapitulate the structural and functional characteristics of the kidneys, providing a promising model to study mitochondria-related kidney physiology and disease in a personalized manner.

CyTOF analysis identifies unusual immune cells in urine of BCG-treated bladder cancer patients.

High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although most bladder cancer patients respond well to BCG, there is no clinical parameter predictive of treatment response, and when treatment fails, the prognosis is very poor. Further, a high percentage of NMIBC patients treated with BCG suffer unwanted effects that force them to stop treatment. Thus, early identification of patients in which BCG treatment will fail is really important. Here, to identify early stage non-invasive biomarkers of non-responder patients and patients at risk of abandoning the treatment, we longitudinally analysed the phenotype of cells released into the urine of bladder cancer patients 3-7 days after BCG instillations. Mass cytometry (CyTOF) analyses revealed a large proportion of granulocytes and monocytes, mostly expressing activation markers. A novel population of CD15+CD66b+CD14+CD16+ cells was highly abundant in several samples; expression of these markers was confirmed using flow cytometry and qPCR. A stronger inflammatory response was associated with increased cell numbers in the urine; this was not due to hematuria because the cell proportions were distinct from those in the blood. This pilot study represents the first CyTOF analysis of cells recruited to urine during BCG treatment, allowing identification of informative markers associated with treatment response for sub-selection of markers to confirm using conventional techniques. Further studies should jointly evaluate cells and soluble factors in urine in larger cohorts of patients to characterise the arms of the immune response activated in responders and to identify patients at risk of complications from BCG treatment.

Urine Cells-derived iPSCs: An Upcoming Frontier in Regenerative Medicine.

There is a momentous surge in the development of stem cell technology, such as therapeutic and diagnostic tools. Stem cell-derived cells are currently used in various clinical trials. However, key issues and challenges faced involve the low differentiation efficiency, integration and functioning of transplanted stem cells-derived cells. Extraction of bone marrow, adipose or other mesenchymal stem cells (MSCs) involves invasive methods, specialized skills and expensive technologies. Urine-derived cells, on the other hand, are obtained by non-invasive methods; samples can be obtained repeatedly from patients of any age. Urine-derived cells are used to generate reprogrammed or induced pluripotent stem cells (iPSCs) which can be cultured and differentiated into various types of cell lineages for biomedical investigations and drug testing in vitro or in vivo using model animals of human diseases. Urine cells-derived iPSCs (UiPSCs) have emerged as a major area of research having immense therapeutic significance. Given that preliminary preclinical studies are successful in terms of safety and as a regenerative tool, the UiPSCs will pave the way to the development of various types of autologous stem cell therapies.

An Overview on Promising Somatic Cell Sources Utilized for the Efficient Generation of Induced Pluripotent Stem Cells.

Human induced Pluripotent Stem Cells (iPSCs) have enormous potential in understanding developmental biology, disease modeling, drug discovery, and regenerative medicine. The initial human iPSC studies used fibroblasts as a starting cell source to reprogram them; however, it has been identified to be a less appealing somatic cell source by numerous studies due to various reasons. One of the important criteria to achieve efficient reprogramming is determining an appropriate starting somatic cell type to induce pluripotency since the cellular source has a major influence on the reprogramming efficiency, kinetics, and quality of iPSCs. Therefore, numerous groups have explored various somatic cell sources to identify the promising sources for reprogramming into iPSCs with different reprogramming factor combinations. This review provides an overview of promising easily accessible somatic cell sources isolated in non-invasive or minimally invasive manner such as keratinocytes, urine cells, and peripheral blood mononuclear cells used for the generation of human iPSCs derived from healthy and diseased subjects. Notably, iPSCs generated from one of these cell types derived from the patient will offer ethical and clinical advantages. In addition, these promising somatic cell sources have the potential to efficiently generate bona fide iPSCs with improved reprogramming efficiency and faster kinetics. This knowledge will help in establishing strategies for safe and efficient reprogramming and the generation of patient-specific iPSCs from these cell types.

Identification of New Transcription Factors that Can Promote Pluripotent Reprogramming.

BACKGROUND: Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered. METHODS: The chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro. RESULTS: ATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes. CONCLUSIONS: In conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease.

Application of urine cells in drug intervention for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a lethal childhood neurodegenerative disorder that is caused by the homozygous deletion of survival motor neuron 1 (SMN1). To date, no effective treatments are available. In the current study, urine cells taken from SMA patients were cultured and the application of patient-derived urine cells was determined in drug intervention. A total of 13 SMA patient-derived urine cell lines and 40 control cell lines were established. SMN was highly expressed in the nucleus and cytoplasm. Patient-derived urine cells expressed low levels of SMN protein compared with controls, they exhibited good tolerance to chemical and electrical damage. SMN expression was upregulated following treatment with histone deacetylase inhibitors and the effect was greater in groups treated with morpholino modified antisense oligo, which targets ISS-N1 in SMN2 intron 7. The results of the current study indicated that SMA patient-derived urine cells may be useful in the initial screening of potential compounds and drugs to treat SMA.

Urine-derived induced pluripotent stem cells as a modeling tool to study rare human diseases.

Rare diseases with a low prevalence are a key public health issue because the causes of those diseases are difficult to determine and those diseases lack a clearly established or curative treatment. Thus, investigating the molecular mechanisms that underlie the pathology of rare diseases and facilitating the development of novel therapies using disease models is crucial. Human induced pluripotent stem cells (iPSCs) are well suited to modeling rare diseases since they have the capacity for self-renewal and pluripotency. In addition, iPSC technology provides a valuable tool to generate patient-specific iPSCs. These cells can be differentiated into cell types that have been affected by a disease. These cells would circumvent ethical concerns and avoid immunological rejection, so they could be used in cell replacement therapy or regenerative medicine. To date, human iPSCs could have been generated from multiple donor sources, such as skin, adipose tissue, and peripheral blood. However, these cells are obtained via invasive procedures. In contrast, several groups of researchers have found that urine may be a better source for producing iPSCs from normal individuals or patients. This review discusses urinary iPSC (UiPSC) as a candidate for modeling rare diseases. Cells obtained from urine have overwhelming advantages compared to other donor sources since they are safely, affordably, and frequently obtained and they are readily obtained from patients. The use of iPSC-based models is also discussed. UiPSCs may prove to be a key means of modeling rare diseases and they may facilitate the treatment of those diseases in the future.

Mitochondrial Disease-Specific Induced Pluripotent Stem Cell Models: Generation and Characterization.

Mitochondrial disease is a group of disorders caused by dysfunctional mitochondria, of which the mutation in the mitochondrial DNA is one of the primary factors. However, the molecular pathogenesis of mitochondrial diseases remains poorly understood due to lack of cell models. Patient-specific induced pluripotent stem cells (iPS cells or iPSCs) are originated from individuals suffering different diseases but carrying unchanged disease causing gene. Therefore, patient-specific iPS cells can be used as excellent cell models to elucidate the mechanisms underlying mitochondrial diseases. Here we present a detailed protocol for generating iPS cells from urine cells and fibroblasts for instance, as well as a series of characterizations.

Direct reprogramming of urine-derived cells with inducible MyoD for modeling human muscle disease.

BACKGROUND: Cellular models of muscle disease are taking on increasing importance with the large number of genes and mutations implicated in causing myopathies and the concomitant need to test personalized therapies. Developing cell models relies on having an easily obtained source of cells, and if the cells are not derived from muscle itself, a robust reprogramming process is needed. Fibroblasts are a human cell source that works well for the generation of induced pluripotent stem cells, which can then be differentiated into cardiomyocyte lineages, and with less efficiency, skeletal muscle-like lineages. Alternatively, direct reprogramming with the transcription factor MyoD has been used to generate myotubes from cultured human fibroblasts. Although useful, fibroblasts require a skin biopsy to obtain and this can limit their access, especially from pediatric populations. RESULTS: We now demonstrate that direct reprogramming of urine-derived cells is a highly efficient and reproducible process that can be used to establish human myogenic cells. We show that this method can be applied to urine cells derived from normal individuals as well as those with muscle diseases. Furthermore, we show that urine-derived cells can be edited using CRISPR/Cas9 technology. CONCLUSIONS: With progress in understanding the molecular etiology of human muscle diseases, having a readily available, noninvasive source of cells from which to generate muscle-like cells is highly useful.

Footprint- and xeno-free human iPSCs derived from urine cells using extracellular matrix-based culture conditions.

The efficient generation of integration- and xeno-free iPSCs is a prerequisite for their use in clinical applications. Furthermore, non-invasiveness of somatic cell acquisition for iPSC generation is another factor to consider. In this study, we established a practical, simple, and convenient method to generate integration- and xeno-free iPSCs from urine cells which can be obtained in a non-invasive manner. Our method was based on extracellular matrix-based xeno-free iPSC culture condition and episomal transfection, and worked efficiently with both urine cells and adipose-derived stromal cells (ADSCs). To obtain strictly xeno-free iPSCs, we also formulated a new xeno-free culture medium for primary urine cells. Intriguingly, urine cells displayed slower growth, and more dramatic increase in apoptosis at high passage numbers than ADSCs. However, urine cells at low passage (

The consequence of delayed fixation on subsequent preservation of urine cells.

OBJECTIVES: Degenerative changes caused by delays in urine preservation contribute to false-negative and false-positive interpretation of urothelial disease in cytology. The aim of this study is to assess whether the delay of fixation of urine samples makes any significant difference to urine cytology and morphology, and the limit of acceptability of delay for routine use in the hospital laboratory. METHODS: Three cell collection fluids were evaluated by analyzing the preservation and degeneration of cells in urine samples. In this study, 50 voided urine specimens were taken at random from females complaining of vaginal discharge. Each specimen was divided into three sterile containers. The first was immediately centrifugated and the deposit was smeared onto a cleaned micro slide and immediately fixed into 95% ethyl alcohol for 15 minutes. The remaining two were prepared in the same manner, however, the second after two hours of collection and the third after four hours of collection. The degree of degeneration and thus the preservation were assessed by a table of chosen criteria, then ranked and analyzed using Friedman's nonparametric test, at p=0.05. RESULTS: The results showed a significant difference between the preservation and the delay in urine fixation, p<0.0001. CONCLUSION: Any delay in fixation of urine specimen for cytology affects the preservation of cells, which may result in miss diagnosis. It is recommended that urine samples for cytology should be fixed immediately after collection.