Reactivation of NCAM1 Defines a Subpopulation of Human Adult Kidney Epithelial Cells with Clonogenic and Stem/Progenitor Properties
2013; Elsevier BV; Volume: 183; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2013.07.034
ISSN1525-2191
AutoresElla Buzhor, Dorit Omer, Orit Harari‐Steinberg, Zohar Dotan, Einav Vax, Sara Pri‐Chen, Sally Metsuyanim, Oren Pleniceanu, Ronald S. Goldstein, Benjamin Dekel,
Tópico(s)Tissue Engineering and Regenerative Medicine
ResumoThe nephron is composed of a monolayer of epithelial cells that make up its various compartments. In development, these cells begin as mesenchyme. NCAM1, abundant in the mesenchyme and early nephron lineage, ceases to express in mature kidney epithelia. We show that, once placed in culture and released from quiescence, adult human kidney epithelial cells (hKEpCs), uniformly positive for CD24/CD133, re-express NCAM1 in a specific cell subset that attains a stem/progenitor state. Immunosorted NCAM1+ cells overexpressed early nephron progenitor markers (PAX2, SALL1, SIX2, WT1) and acquired a mesenchymal fate, indicated by high vimentim and reduced E-cadherin levels. Gene expression and microarray analysis disclosed both a proximal tubular origin of these cells and molecules regulating epithelial–mesenchymal transition. NCAM1+ cells generated clonal progeny when cultured in the presence of fetal kidney conditioned medium, differentiated along mesenchymal lineages but retained the unique propensity to generate epithelial kidney spheres and produce epithelial renal tissue on single-cell grafting in chick CAM and mouse. Depletion of NCAM1+ cells from hKEpCs abrogated stemness traits in vitro. Eliminating these cells during the regenerative response that follows glycerol-induced acute tubular necrosis worsened peak renal injury in vivo. Thus, higher clone-forming and developmental capacities characterize a distinct subset of adult kidney-derived cells. The ability to influence an endogenous regenerative response via NCAM1 targeting may lead to novel therapeutics for renal diseases. The nephron is composed of a monolayer of epithelial cells that make up its various compartments. In development, these cells begin as mesenchyme. NCAM1, abundant in the mesenchyme and early nephron lineage, ceases to express in mature kidney epithelia. We show that, once placed in culture and released from quiescence, adult human kidney epithelial cells (hKEpCs), uniformly positive for CD24/CD133, re-express NCAM1 in a specific cell subset that attains a stem/progenitor state. Immunosorted NCAM1+ cells overexpressed early nephron progenitor markers (PAX2, SALL1, SIX2, WT1) and acquired a mesenchymal fate, indicated by high vimentim and reduced E-cadherin levels. Gene expression and microarray analysis disclosed both a proximal tubular origin of these cells and molecules regulating epithelial–mesenchymal transition. NCAM1+ cells generated clonal progeny when cultured in the presence of fetal kidney conditioned medium, differentiated along mesenchymal lineages but retained the unique propensity to generate epithelial kidney spheres and produce epithelial renal tissue on single-cell grafting in chick CAM and mouse. Depletion of NCAM1+ cells from hKEpCs abrogated stemness traits in vitro. Eliminating these cells during the regenerative response that follows glycerol-induced acute tubular necrosis worsened peak renal injury in vivo. Thus, higher clone-forming and developmental capacities characterize a distinct subset of adult kidney-derived cells. The ability to influence an endogenous regenerative response via NCAM1 targeting may lead to novel therapeutics for renal diseases. Kidney disease is a major worldwide health burden. Given the limited number of treatments currently available, discovering novel ways to stimulate kidney repair is an important therapeutic goal. Many adult tissues (such as skin, the hematopoietic system, and the intestine) are considered to harbor cells that self-renew and differentiate to form clones of stem, progenitor, and mature cells of the organ, fitting within the criteria of tissue-specific multipotential stem cells.1Weissman I. The ISSCR: who are we and where are we going?.Cell Stem Cell. 2009; 5: 151-153Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 2Barker N. van de Wetering M. Clevers H. The intestinal stem cell.Genes Dev. 2008; 22: 1856-1864Crossref PubMed Scopus (475) Google Scholar, 3Blanpain C. Fuchs E. Epidermal homeostasis: a balancing act of stem cells in the skin.Nat Rev Mol Cell Biol. 2009; 10: 207-217Crossref PubMed Scopus (869) Google Scholar, 4Kondo M. Wagers A.J. Manz M.G. Prohaska S.S. Scherer D.C. Beilhack G.F. Shizuru J.A. Weissman I.L. Biology of hematopoietic stem cells and progenitors: implications for clinical application.Annu Rev Immunol. 2003; 21: 759-806Crossref PubMed Scopus (783) Google Scholar In contrast to these rapidly cycling organs, parenchymal cells of the kidney are considered to be mostly static under steady-state conditions and can be induced to divide only under very specific conditions, limiting the overall regenerative capacity of the nephron, the kidney's functional unit.5Little M.H. Regrow or repair: potential regenerative therapies for the kidney.J Am Soc Nephrol. 2006; 17: 2390-2401Crossref PubMed Scopus (140) Google Scholar All nephron epithelia arise from a self-renewing nephron progenitor population that resides in the metanephric mesenchyme of the developing kidney's nephrogenic zone, specifically in the condensed mesenchyme that interacts with the ureteric bud, the precursor for the collecting system and undergoes mesenchymal–epithelial transition (MET). With the completion of mammalian nephrogenesis (gestational week 34 in humans, 2 postnatal weeks in mice), this undifferentiated nephron-forming progenitor population is entirely exhausted. Therefore, in contrast to fish,6Diep C.Q. Ma D. Deo R.C. Holm T.M. Naylor R.W. Arora N. Wingert R.A. Bollig F. Djordjevic G. Lichman B. Zhu H. Ikenaga T. Ono F. Englert C. Cowan C.A. Hukriede N.A. Handin R.I. Davidson A.J. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish.Nature. 2011; 470: 95-100Crossref PubMed Scopus (218) Google Scholar no progenitor population with nephrogenic potential similar to the metanephric mesenchyme or condensed mesenchyme physiologically exists in the mammalian adult kidney.7Hartman H.A. Lai H.L. Patterson L.T. Cessation of renal morphogenesis in mice.Dev Biol. 2007; 310: 379-387Crossref PubMed Scopus (236) Google Scholar, 8Pleniceanu O. Harari-Steinberg O. Dekel B. Concise review: kidney stem/progenitor cells: differentiate, sort out, or reprogram?.Stem Cells. 2010; 28: 1649-1660Crossref PubMed Scopus (76) Google Scholar Using microarrays, we have previously characterized the renal epithelial progenitor population of the developing human kidney.9Dekel B. Metsuyanim S. Schmidt-Ott K.M. Fridman E. Jacob-Hirsch J. Simon A. Pinthus J. Mor Y. Barasch J. Amariglio N. Reisner Y. Kaminski N. Rechavi G. Multiple imprinted and stemness genes provide a link between normal and tumor progenitor cells of the developing human kidney.Cancer Res. 2006; 66: 6040-6049Crossref PubMed Scopus (114) Google Scholar Our results showed expression of a set of genes, including transcription factors that specify nephron lineage and are considered nephron progenitor markers,10Dressler G.R. The cellular basis of kidney development.Annu Rev Cell Dev Biol. 2006; 22: 509-529Crossref PubMed Scopus (480) Google Scholar, 11Rosenblum N.D. Developmental biology of the human kidney.Semin Fetal Neonatal Med. 2008; 13: 125-132Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar a polycomb group, and Wnt pathway molecules concomitant with several surface antigens such as NCAM1. After validation by fluorescence-activated cell sorting (FACS) analysis, we demonstrated the usefulness of NCAM1 for immunoselection strategies and cell sorting of specific human developmental renal progenitor subsets.9Dekel B. Metsuyanim S. Schmidt-Ott K.M. Fridman E. Jacob-Hirsch J. Simon A. Pinthus J. Mor Y. Barasch J. Amariglio N. Reisner Y. Kaminski N. Rechavi G. Multiple imprinted and stemness genes provide a link between normal and tumor progenitor cells of the developing human kidney.Cancer Res. 2006; 66: 6040-6049Crossref PubMed Scopus (114) Google Scholar, 12Metsuyanim S. Harari-Steinberg O. Buzhor E. Omer D. Pode-Shakked N. Ben-Hur H. Halperin R. Schneider D. Dekel B. Expression of stem cell markers in the human fetal kidney.PloS One. 2009; 4: e6709Crossref PubMed Scopus (98) Google Scholar, 13Harari-Steinberg O. Matsuyanim S. Omer D. Gnatek Y. Gershon R. Pri-Chen S. Ozdemir D.D. Lerenthal Y. Noiman T. Ben-Hur H. Vaknin Z. Schneider D.F. Aronow B.J. Goldstein R.S. Hohenstein P. Indentification of human nephron progenitors capable of generation of kidney structures and functional repair of chronic renal disease.EMBO Mol Med. 2013; ([Epub ahead of print])http://doi.dx.org/10.1002/emmm.201201584Google Scholar After cessation of nephrogenesis, the early nephron progenitor markers are down-regulated in both murine kidneys7Hartman H.A. Lai H.L. Patterson L.T. Cessation of renal morphogenesis in mice.Dev Biol. 2007; 310: 379-387Crossref PubMed Scopus (236) Google Scholar and human8Pleniceanu O. Harari-Steinberg O. Dekel B. Concise review: kidney stem/progenitor cells: differentiate, sort out, or reprogram?.Stem Cells. 2010; 28: 1649-1660Crossref PubMed Scopus (76) Google Scholar kidneys. Similarly, NCAM1, which is strongly expressed in the condensed mesenchyme, nephrogenic zone, and in Wilms' tumor progenitor blastema, is not expressed in mature kidney epithelia after nephron differentiation.12Metsuyanim S. Harari-Steinberg O. Buzhor E. Omer D. Pode-Shakked N. Ben-Hur H. Halperin R. Schneider D. Dekel B. Expression of stem cell markers in the human fetal kidney.PloS One. 2009; 4: e6709Crossref PubMed Scopus (98) Google Scholar, 13Harari-Steinberg O. Matsuyanim S. Omer D. Gnatek Y. Gershon R. Pri-Chen S. Ozdemir D.D. Lerenthal Y. Noiman T. Ben-Hur H. Vaknin Z. Schneider D.F. Aronow B.J. Goldstein R.S. Hohenstein P. Indentification of human nephron progenitors capable of generation of kidney structures and functional repair of chronic renal disease.EMBO Mol Med. 2013; ([Epub ahead of print])http://doi.dx.org/10.1002/emmm.201201584Google Scholar, 14Pode-Shakked N. Metsuyanim S. Rom-Gross E. Mor Y. Fridman E. Goldstein I. Amariglio N. Rechavi G. Keshet G. Dekel B. Developmental tumourigenesis: NCAM as a putative marker for the malignant renal stem/progenitor cell population.J Cell Mol Med. 2009; 13: 1792-1808Crossref PubMed Scopus (73) Google Scholar, 15Pode-Shakked N. Shukrun R. Mark-Danieli M. Tsvetkov P. Bahar S. Pri-Chen S. Goldstein R.S. Rom-Gross E. Mor Y. Fridman E. Meir K. Simon A. Magister M. Kaminski N. Goldmacher V.S. Harari-Steinberg O. Dekel B. The isolation and characterization of renal cancer initiating cells from human Wilms' tumour xenografts unveils new therapeutic targets.EMBO Mol Med. 2013; 5: 18-37Crossref PubMed Scopus (66) Google Scholar Here, we show that isolates of human kidney epithelia grown under adherent conditions proliferate and activate NCAM1 in a specific cell subset showing early renal stem/progenitor characteristics and function. The in vitro identification of distinct NCAM1+ clone-forming cells and the possible beneficial role of NCAM1+ cells in regenerating kidney epithelia in vivo suggest NCAM1 as a target for manipulation for an enhanced regenerative response. Normal human adult kidney samples were retrieved from borders of renal cell carcinoma tumors from patients undergoing partial or total nephrectomy at Sheba Medical Center and Wolfson Hospital. This procedure was performed after approval by the local ethical committee and signed informed consent from the patient. The samples were minced in Hanks' balanced salt solution, soaked in collagenase for 2 hours, and then cultured in serum-containing medium (SCM), which consisted of Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 1% l-glutamine, 1% penicillin–streptomycin, and the following growth factors: 50 ng/mL bFGF, 50 ng/mL EGF, and 5 ng/mL SCF (R&D Systems, Minneapolis, MN). Serum-free medium (SFM) consisted of 500 mL Dulbecco's modified Eagle's medium/F12 (DMEM/F12, 1:1; Life Technologies, Carlsbad, CA), 1% penicillin–streptomycin, 2 mL B27 supplement (Life Technologies), 4 μg/mL heparin, 1% nonessential amino acids (Life Technologies), 1% sodium pyruvate (Life Technologies), 1% l-glutamine, 1 mL lipid mix (Sigma-Aldrich, St. Louis, MO), 5 mL 100× N2 supplement (Life Technologies), 5 mL growth factor mix [200 mL of growth factor mix containing 100 mL DMEM/F12, 4 mL 30% glucose, 200 mg transferrin, 50 mg insulin in 20 mL of water, 19.3 mg putrescine in 20 mL distilled water, 200 μL sodium selenate (0.3 mmol/L stock), 20 μL progesterone (2 mmol/L stock)], 10 ng/mL FGF, and 20 ng/mL EGF. Sphere formation was tested by seeding the cells in polyHEMA (Sigma-Aldrich) precoated plates in SFM. Fetal kidney conditioned medium (FKCM) was obtained by combining SCM and supernatants from fetal kidney cultures (cultured in SCM) of passages 1 to 3 at a 1:1 ratio. Monolayer cells were detached from culture plates with 0.25% trypsin (Life Technologies). Viable cell number was determined using Trypan Blue staining (Life Technologies). In magnetic-activated cell sorting (MACS), CD56 (NCAM1) microbeads (Miltenyi Biotec, Auburn, CA) were used for single-marker cell separation. Positive and negative fractions were separated using Mini or Midi MACS columns (Miltenyi Biotec), according to the manufacturer's protocol. In FACS sorting, cells were sorted with anti-NCAM–phycoerythrin (PE) (eBioscience, San Diego, CA) using a FACSAria fluorescence-activated cell sorter and FACSDiva software version 4.0 (BD Biosciences, San Jose, CA), as described previously.14Pode-Shakked N. Metsuyanim S. Rom-Gross E. Mor Y. Fridman E. Goldstein I. Amariglio N. Rechavi G. Keshet G. Dekel B. Developmental tumourigenesis: NCAM as a putative marker for the malignant renal stem/progenitor cell population.J Cell Mol Med. 2009; 13: 1792-1808Crossref PubMed Scopus (73) Google Scholar Quantitative real-time RT-PCR (RT-qPCR) reactions were performed as described previously.12Metsuyanim S. Harari-Steinberg O. Buzhor E. Omer D. Pode-Shakked N. Ben-Hur H. Halperin R. Schneider D. Dekel B. Expression of stem cell markers in the human fetal kidney.PloS One. 2009; 4: e6709Crossref PubMed Scopus (98) Google Scholar, 16Buzhor E. Harari-Steinberg O. Omer D. Metsuyanim S. Jacob-Hirsch J. Noiman T. Dotan Z. Goldstein R.S. Dekel B. Kidney spheroids recapitulate tubular organoids leading to enhanced tubulogenic potency of human kidney-derived cells.Tissue Eng Part A. 2011; 17: 2305-2319Crossref PubMed Scopus (55) Google Scholar In brief, total RNA from cells was isolated using an RNeasy micro kit (Qiagen, Hilden, Germany; Valencia, CA), according to the manufacturer's instructions. cDNA was synthesized using a high-capacity cDNA reverse-transcription kit (Life Technologies) on total RNA. qPCR was performed using an ABI 7900HT sequence detection system (Life Technologies) in the presence of TaqMan gene expression master mix (Life Technologies), and PCR amplification was performed using gene-specific TaqMan gene expression assay premade kits. Each analysis reaction was performed in duplicate or triplicate. GAPDH and HPRT1 were used as endogenous control throughout all experiments. Analysis was performed using the ΔΔCT method, which determines fold change in gene expression relative to a comparator sample. PCR results were analyzed using SDS RQ Manager software version 1.2 (Life Technologies). Limiting dilution assay was performed on total human adult kidney or separated NCAM1+ versus NCAM1− cell fractions. In brief, sorted cells were plated in 96-well plates (Greiner Bio-One, Frickenhausen, Germany) in 150 μL of culture medium, at 1 or 5 cells per well. The number of colonized wells was recorded after 2 to 4 weeks. Confluent viable clones of total hKEpCs were isolated and propagated. NCAM1+ and NCAM1− cell fractions were subjected to CellTiter 96 AQ nonradioactive cell proliferation (Promega, Madison, WI), based on the novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), according to the manufacturer's protocol. Wavelength for optical density is 450 nm. Osteogenic differentiation of NCAM1+ cells was performed in NH OsteoDiff medium (Miltenyi Biotec), according to the manufacturer's protocol. NCAM1+ cells were seeded at a concentration of 7.5 × 103 cells per well (1 mL medium per well) in 24-well plates and were incubated for 10 days. Osteoblast detection was performed by staining for alkaline phosphatase activity with an alkaline phosphatase kit (Sigma-Aldrich), according to the manufacturer's protocol. Adipogenic differentiation of NCAM1+ cells was performed in NH AdipoDiff medium (Miltenyi Biotec), according to the manufacturer's protocol. NCAM1+ cells were seeded at a concentration of 12.5 × 103 cells per well (1 mL medium per well) in 24-well plates and were incubated for 21 days. Adipocyte detection was performed by staining with Oil Red O solution (Sigma-Aldrich). DMEM was used as a control for differentiation assessment in both assays. Human adult renal NCAM1+ and NCAM1− cell fractions obtained from one adult donor were evaluated using Affymetrix HU GENE1.0ORD oligonucleotide arrays (Affymetrix technical note: Data sheet: GeneChip HuGene 1.0 ST Array System for Human, Mouse and Rat. Santa Clara, CA). Total RNA from each sample was used to prepare biotinylated target DNA, according to the manufacturer's recommendations. Target cDNA generated from each sample was processed using an Affymetrix Gene Chip instrument system [Affymetrix technical note: User Manual: GeneChip Whole Transcript (WT) Sense Target Labeling Assay. Santa Clara, CA]. The quality and amount of starting RNA was confirmed using agarose gel or by use of an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). When scanning was done, array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches on the chip surface. The signals derived from the array were assessed using various quality assessment metrics. Details of quality control are provided in the Affymetrix data sheet cited above. Gene-level RMA sketch algorithm [Affymetrix Expression Console and Partek Genomics Suite version 6.2 (Partek, St. Louis, MO)] was used for generation of crude data. Significantly changed genes were filtered as changed by at least twofold (P = 0.05). Genes were filtered and analyzed using unsupervised and supervised hierarchical cluster analysis [Partek Genomics Suite and Spotfire DecisionSite for Functional Genomics version 9.1.2 (TIBCO Spotfire, Somerville, MA)] to get a first assessment of the data. Further processing included functional analysis and over-representation calculations based on Gene Ontology and using the publicly available Database for Annotation, Visualization and Integrated Discovery and associated tools (DAVID tools version 6.7; http://david.abcc.ncifcrf.gov). Over-representation calculations were performed using the DAVID Ease tool, according to the Affymetrix technical notes cited above. Ingenuity Pathway Analysis software (IPA version 7; Ingenuity Systems, Redwood City, CA) was used for network analysis. The microarray data were deposited with the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession no. GSE49100). Tubular segments were identified using the segment-specific markers. Proximal tubules were identified with biotinylated Lotus tetragonolobus lectin (LTA) (1:200, Vector Laboratories) and aminopeptidase A (ENPEP) (1:75; Sigma-Aldrich); distal tubules and collecting ducts were identified with biotinylated Dolichos biflorus agglutinin (DBA) (1:200; Vector Laboratories); and epithelial cells were identified with cytokeratin (1:250; Dako). Before staining, cells were fixed in ice-cold 95% ethanol–5% acetic acid for 10 minutes, washed in PBS, and blocked with 0.1% bovine serum albumin in PBS for 1 hour. Detection was performed with streptavidin Alexa Fluor 488 (1:1000, Jackson ImmunoResearch Laboratories) and anti-rabbit Alexa Fluor 488 (1:1000; Life Technologies). Slides were counterstained with ProLong Gold antifade reagent with DAPI (Life Technologies). Photomicrographs were made on a Nikon TI-E inverted microscope. Lorvotuzumab mertansine (alias huN901-DM1 or IMGN901) is a humanized version of the anti-CD56 antibody N901, conjugated to the highly cytotoxic maytansine derivative DM1 via a hindered disulfide linker17Wang L. Amphlett G. Blättler W.A. Lambert J.M. Zhang W. Structural characterization of the maytansinoid–monoclonal antibody immunoconjugate, huN901–DM1, by mass spectrometry.Protein Sci. 2005; 14: 2436-2446Crossref PubMed Scopus (287) Google Scholar (ImmunoGen, Waltham, MA). hKEpCs were cultured at concentrations of 500, 1000, 2000, and 4000 cells per well in 96-well plates. After 1, 3, 5, and 7 days in culture, cell proliferation was evaluated using the MTS cell proliferation assay (Promega). Cell plating concentration (cells per well) for the huN901-DM1 assays was based on a sufficient amount of cells at the beginning of the experiment so as not to reach confluence (and therefore massive cell death) by the end of the experiment. To determine the LD50 of huN901-DM1 for hKEpCs, cells were seeded in 96-well plates at 104 cells per well for 24 hours in growth medium, which was then replaced with medium containing the conjugate in a range of concentrations between 1.6 nmol/L and 1.675 μmol/L, with medium alone serving as a control. After a 5-day incubation period, cell survival was assessed by the MTS proliferation assay and LD50 was determined. hKEpCs were seeded at 2 × 104 cells per well in six-well plates in duplicate and were treated with anti-NCAM antibody (huN901), anti-NCAM antibody conjugated with immunotoxin (huN901-DM1; 0.1 μmol/L), or control. On day 5 of treatment, cells from all groups were subjected to limiting dilution, sphere formation, FACS, and MTS assays. Grafting of hKEpC NCAM1+ cells on the chick embryo CAM was performed as described previously.16Buzhor E. Harari-Steinberg O. Omer D. Metsuyanim S. Jacob-Hirsch J. Noiman T. Dotan Z. Goldstein R.S. Dekel B. Kidney spheroids recapitulate tubular organoids leading to enhanced tubulogenic potency of human kidney-derived cells.Tissue Eng Part A. 2011; 17: 2305-2319Crossref PubMed Scopus (55) Google Scholar, 18Noiman T. Buzhor E. Metsuyanim S. Harari-Steinberg O. Morgenshtern C. Dekel B. Goldstein R.S. A rapid in vivo assay system for analyzing the organogenetic capacity of human kidney cells.Organogenesis. 2011; 7: 140-144Crossref PubMed Scopus (20) Google Scholar In brief, fertile chicken eggs were obtained from a commercial supplier and incubated at 37°C. On day 9 or 10 of incubation, a window was opened in the shell, and the CAM was exposed. hKEp NCAM1+ cells separated by MACS were suspended in 50 μL medium and Matrigel (BD Biosciences) (1:1 by volume) and pipetted into a plastic ring placed on the chorioallantoic membrane (CAM). The egg was then sealed with adhesive tape and returned to the incubator. After 1 week, the graft was removed, paraffin-embedded, and serially sectioned at 6 μm for histological and immunocytochemical analyses, as described previously.16Buzhor E. Harari-Steinberg O. Omer D. Metsuyanim S. Jacob-Hirsch J. Noiman T. Dotan Z. Goldstein R.S. Dekel B. Kidney spheroids recapitulate tubular organoids leading to enhanced tubulogenic potency of human kidney-derived cells.Tissue Eng Part A. 2011; 17: 2305-2319Crossref PubMed Scopus (55) Google Scholar, 18Noiman T. Buzhor E. Metsuyanim S. Harari-Steinberg O. Morgenshtern C. Dekel B. Goldstein R.S. A rapid in vivo assay system for analyzing the organogenetic capacity of human kidney cells.Organogenesis. 2011; 7: 140-144Crossref PubMed Scopus (20) Google Scholar NCAM1+ cells separated by MACS were suspended in 200 μL Matrigel (BD Biosciences) and were injected subcutaneously into NOD/SCID mice (Harlan Laboratories, Israel). At 14 days after injection, the grafts were removed, paraffin-embedded, and serially sectioned at 6 μm for histological and immunocytochemical analyses. Acute kidney injury (AKI) was induced in female NOD/SCID mice by intramuscular injection with 50% hypertonic glycerol (Sigma-Aldrich) solution in water (9 μL/g body weight) after water deprivation for 22 hours. Controlled intramuscular injection of glycerol into the inferior hind limbs was performed under anesthesia (isoflurane inhalation; Abbott Laboratories, North Chicago, IL). Mice received an intravenous injection into the tail vein at 2 hours after glycerol injection, as follows: group 1, saline (n = 8); group 2, NCAM1+ cells (n = 7, 1.5 × 106 cells; n = 3, 0.43 × 106); and group 3, NCAM1− cells (n = 7, 1.5 × 106 cells; n = 3, 0.43 × 106). Human adult kidney cells were obtained from three different patients. Blood samples from mice were collected for blood urea nitrogen (BUN) and creatinine measurements at 3 and 14 days after glycerol injection; the animals were then sacrificed. Animal experiments were performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011), after approval by the Institutional Animal Care and Use Committee. BALB/c mice (Harlan Laboratories) received an intravenous injection into the tail vein 2 hours after glycerol injection, as follows: group 1, saline (n = 10); group 2, huN901-DM1 (18 μg/g; n = 10); and group 3, huN901(18 μg/g; n = 10). Blood samples from mice were collected for creatinine measurements at 3 days after glycerol injection; the animals were then sacrificed. For NCAM1 staining, mouse kidneys were harvested at 3 days after glycerol injection, paraffin-embedded, and serially sectioned at 6 μm for immunofluorescence analysis with anti-NCAM1 antibody (Epitomics, Burlingame, CA). For FACS analysis, injured mouse kidneys were soaked in collagenase for 1 hour and the cells were stained with huN901 and human-IgG–fluorescein isothiocyanate (Abcam, Cambridge, MA). To establish genetically marked hKEpCs, HEK293 cells were initially transformed. HEK293 cells were maintained in DMEM supplemented with 10% fetal calf serum, l-glutamine, penicillin, and streptomycin (Biological Industries, Beit-Ha'emek, Israel), at 37°C in a 5% CO2–enriched atmosphere. Cells were transfected using calcium phosphate with three lentiviral vectors: 7.5 μg pHR-CMV-mCherry, 5 μg ΔR8.2, and 2.5 μg pMD2.G. After 6 hours, the supernatants were replaced with 5 mL of fresh medium. Supernatants of transfected cells were supplemented with HEPES (pH 7.0; 50 mmol/L final concentration) and filtered through a 0.45-μm pore-size filter; 2 mL was placed on the targeted cells for 2 hours with the addition of 8 μg/mL Polybrene (hexadimethrine bromide; Sigma-Aldrich), and then 3 mL of fresh medium was added. These viral-like particles were used to infect hKEpCs (2 × 105 cells in 60-mm-diameter dishes). Expression of the mCherry reporter gene was analyzed at 2 days after infection. mCherry-labeled hKEpCs were injected intravenously into NOD/SCID mice via the tail vein at 2 hours after glycerol-induced AKI. At 24 hours after cell injection, the animals were sacrificed. Both kidneys were surgically removed and were immediately analyzed with a CRi Maestro II in vivo imaging system (Caliper Life Sciences, Hopkinton, MA). Fluorescence images were obtained with an excitation wavelength of 465 nm and emission wavelength range of 500 to 800 nm. RNA was isolated from the dissected kidneys using TRIzol reagent (Life Technologies) and then was reverse-transcribed as described above. Quantitative PCR was performed using a qPCR system for hGAPDH and mβ-actin, using gene-specific TaqMan gene expression assay premade kits (Life Technologies). The PCR products were analyzed by electrophoresis in 2.5% agarose gel and visualized by ethidium bromide staining. Statistical differences between two groups of data were compared by Student's t-test. For all statistical analyses, the level of significance was set as P < 0.05. Except as otherwise indicated, data are expressed as means ± SEM. After the retrieval of a small specimen of human adult kidney tissue from nephrectomized patients, tissue was dissociated into a single-cell suspension and cultured at low densities (approximately 1 cell/cm2) under adherent conditions in T75 flasks, to enhance clonal growth of hKEpCs.16Buzhor E. Harari-Steinberg O. Omer D. Metsuyanim S. Jacob-Hirsch J. Noiman T. Dotan Z. Goldstein R.S. Dekel B. Kidney spheroids recapitulate tubular organoids leading to enhanced tubulogenic potency of human kidney-derived cells.Tissue Eng Part A. 2011; 17: 2305-2319Crossref PubMed Scopus (55) Google Scholar Proliferative hKEpCs comprise several types of kidney epithelia. To determine whether proliferative hKEpCs express NCAM1 after reaching confluence, despite a lack of in situ expression in renal epithelia, we performed FACS analysis and found 15.9 ± 9.1% NCAM1 staining. Previous analysis has shown that putative renal stem-cell surface antigens CD24 and CD133,19Bussolati B. Bruno S. Grange C. Buttiglieri S. Deregibus M.C. Cantino D. Camussi G. Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 20Sagrinati C. Netti G.S. Mazzinghi B. Lazzeri E. Liotta F. Frosali F. Ronconi E. Meini C. Gacci M. Squecco R. Carini M. Gesualdo L. Francini F. Maggi E. Annunziato F. Lasagni L. Serio M. Romagnani S. Romagnani P. Isolation and characterization of multipotent progenitor cells from the Bowman's capsule of adult human kidneys.J Am Soc Nephrol. 2006; 17: 2443-2456Crossref PubMed Scopus (573) Google Scholar as well as the epithelial differentiation marker EpCAM, are widely expressed in proliferative hKEpC12Metsuyanim S. Harari-Steinberg O. Buzhor E. Omer D. Pode-Shakked N. Ben-Hur H. Halperin R. Schneider D. Dekel B. Expression of stem cell markers in the human fetal kidney.PloS One. 2009; 4: e6709Crossref Pu
Referência(s)