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Isolation and Characterization of Nontubular Sca-1⁺Lin^– Multipotent Stem/Progenitor Cells from Adult Mouse Kidney

Benjamin Dekel^*, Lior Zangi^*, Elias Shezen^*, Shlomit Reich-Zeliger^*, Smadar Eventov-Friedman^*, Helena Katchman^*, Jasmin Jacob-Hirsch, Ninette Amariglio, Gideon Rechavi, Raanan Margalit^* and Yair Reisner^*

^* Department of Immunology, Weizmann Institute of Science, Rehovot; and Department of Pediatric Hemato-Oncology and Functional Genomics, Sheba Medical Center, Tel-Hashomer, Israel

Address correspondence to: Dr. Yair Reisner, Weizmann Institute of Science, Department of Immunology, Rehovot, Israel. Phone: +972-8-9344023; Fax: +972-8-9344145; E-mail: yair.reisner{at}weizmann.ac.il

Received for publication February 21, 2005. Accepted for publication August 30, 2006.

	Abstract

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Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Recently, stage-specific embryonic kidney progenitor tissue was used to regenerate nephrons. Through fluorescence-activated cell sorting, microarray analysis, in vitro differentiation assays, mixed lymphocyte reaction, and a model of ischemic kidney injury, this study sought to identify and characterize multipotent organ stem/progenitor cells in the adult kidney. Herein is reported the existence of nontubular cells that express stem cell antigen-1 (Sca-1). This population of small cells includes a CD45-negative fraction that lacks hematopoietic stem cell and lineage markers and resides in the renal interstitial space. In addition, these cells are enriched for 1-integrin, are cytokeratin negative, and show minimal expression of surface markers that typically are found on bone marrow–derived mesenchymal stem cells. Global gene profiling reveals enrichment for many genes downstream of developmental signaling molecules and self-renewal pathways, such as TGF-/bone morphogenic protein, Wnt, or fibroblast growth factor, as well as for those that are involved in specification of mesodermal lineages (myocyte enhancer factor 2A, YY1-associated factor 2, and filamin-). In vitro, they are plastic adherent and slowly proliferating and result in inhibition of alloreactive CD8⁺ T cells, indicative of an immune-privileged behavior. Furthermore, clonal-derived lines can be differentiated into myogenic, osteogenic, adipogenic, and neural lineages. Finally, when injected directly into the renal parenchyma, shortly after ischemic/reperfusion injury, renal Sca-1⁺Lin^– cells, derived from ROSA26 reporter mice, adopt a tubular phenotype and potentially could contribute to kidney repair. These data define a unique phenotype for adult kidney–derived cells, which have potential as stem cells and may contribute to the regeneration of injured kidneys.

	Introduction

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Regenerative medicine is focused on the development of cells, tissues, and organs for the purpose of restoring function through transplantation (1). In this regard, the use of stem cells offers new and powerful strategies for future tissue development and engineering. Stem cells are defined by two major criteria: Pluripotentiality and self-renewal capacity. Perhaps the most characterized stem cell is one that resides in the adult bone marrow (BM), that is, the hematopoietic stem cell (HSC), which gives rise to all blood cell types (2). In addition, mesenchymal stem cells (MSC) are multipotent cells that can be isolated from adult BM and several other tissues and induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, BM stroma, and muscle (3). Recently, it was suggested that BM-derived stem cells can cross boundaries and give rise to a broader array of differentiated cell types, that is, turning blood into liver, brain, pancreas, skin, intestine, and eventually kidney (4–11). However, this phenomenon remains controversial for several reasons. First, several studies cast doubt on the biologic significance and even the existence of such transdifferentiation (12,13). Second, some other investigations that do show a donor cell phenotype in parenchymal cells after transplantation of BM-derived stem cells suggest that it occurs through cell fusion and not by transdifferentiation and generation of cells de novo (14–16). Therefore, the term "regeneration" is used erroneously in this context and should be replaced by "reparative," if indeed that is shown to be the result of stem cell fusion. Third, whether by transdifferentiation or cell fusion, the efficiency of these processes under basal conditions and even when inflicting tissue injury is low, underscoring the functional capacity of adult BM-derived stem cells. For instance, in the kidney, several studies have shown that BM-derived cells adopt the phenotype of proximal tubular cells during acute tubular injury (10,11), as well as that of glomerular endothelial and mesangial cells during glomerular injury (17–19), but only a few report on mild improvement in kidney function (10).

These obstacles emphasize the need to seek renal progenitor cells beyond the few known extrarenal sources. Recent studies have attempted to isolate and characterize tissue stem cells by identifying organ cells that display specific stem cell phenotypes. One such strategy is to identify organ cells that express stem cell antigens (20–23). Of interest are those that were used previously to define murine BM-derived hematopoietic stem cells, primarily stem cell antigen-1 (Sca-1), c-kit, and CD34 (2). Sca-1 is a member of the Ly-6 family and first was reported as one of the early cell surface markers of hematopoietic stem cells (2). Recent reports have demonstrated that multipotential stem cells that were derived from BM and skeletal muscle express Sca-1 (3,21,22,24,25). Moreover, it was shown recently that beyond its role as a marker for muscle progenitors, Sca-1 has an active function in myogenesis (26). Nevertheless, this approach might be complicated by the presence of Sca-1 on mature cells, as shown for medullary renal tubular epithelium and pulmonary vascular endothelium (27,28).

Recently, we showed that early embryonic human and porcine renal precursors remarkably can grow, differentiate, and undergo vascularization, achieving successful organogenesis of urine-producing miniature kidneys (29). It is interesting that cells that reside in these embryonic progenitor tissues exhibit the potential to differentiate, after transplantation or in vitro manipulation, into other professional cell types, in addition to renal epithelia, that participate in kidney organogenesis (myofibroblasts, smooth muscle, and endothelium), as well as nonrenal derivatives, including cartilage, bone, and blood (29–32). These studies suggested the presence of multipotent embryonic renal stem cells. Unfortunately, although highly proliferative, they can be used only in the allogeneic setting, whereas adult cells represent an autologous source.

Here we identified a population of nontubular Sca-1⁺Lin^– cells in the adult kidney, which are distinct from HSC and their progeny. Additional in vitro and in vivo studies suggest that the cells of this fraction are putative kidney-derived stem cells.

	Materials and Methods

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Animals
The mice used were 8 to 12 wk of age. C57BL/6, SJL, CB6F1, and ROSA26 mice were obtained from the Weizmann Institute Animal Center (Rehovot, Israel). DBA/2 mice were obtained from the Roscoe B. Jackson Memorial Laboratory (Bar Harbor, ME). A breeding pair of transgenic H-2^b mice that expressed the T cell receptor (TCR) from the CTL clone 2C with specificity for H-2L^d was provided by J. Nikolic-Zugic (Sloan-Kettering Institute, New York, NY). All protocols were approved by the Institutional Animal Care and Use Committee of the Weizmann Institute of Science.

Isolation and Characterization of Sca-1⁺ Cells from the Adult Murine Kidney
Kidneys of adult (10- to 12-wk-old) C57BL/6 mice were washed extensively with sterile PBS to remove contaminating debris and red blood cells (RBC). Kidneys then were diced and treated with 0.075% collagenase (type D; Sigma-Aldrich, St. Louis, MO) diluted in PBS for 10 min at 37°C with gentle agitation. The collagenase was inactivated with an equal volume of culture medium (DMEM/10% FCS/1% penicillin streptomycin), and the dissolved tissue was minced further and centrifuged for 10 min at low speed. The cellular pellet was resuspended in culture medium and sequentially filtered through 70- and 40-µm mesh filters to remove debris and cell segments. Cell suspensions were treated with cold ACK buffer (0.15 M potassium-ammonium chloride buffer) to remove remaining RBC. A comparison of kidney cells, obtained through mincing and filtration, with or without collagenase, yielded similar results.

Enrichment of Sca-1⁺ cells was achieved by incubating cells with anti–Sca-1 microbeads (Miltenyi Biotec, Auburn, CA) and purification by at least two cycles of magnetic selection (20). Sorted populations were reanalyzed by flow cytometry, and the purity of Sca-1⁺ cells was confirmed before use. FACS analysis was performed using a modified FACScan (BD Biosciences, Mountain View, CA). Fluorescence data were collected using three-decade logarithmic amplification on 25 to 50 x 10³ viable cells, as determined by forward light scatter intensity. Cells were labeled with Sca-1-PE, Sca-1-APC, Sca-1-Biotin; CD45-, B220-, Mac-1 (CD11b)-, NK-, TER119-, CD11c-, CD29-, I-AD-, Fas (CD95)-, and H-2^b-FITC (BD Pharmingen, San Diego, CA); CD34-, CD31-, CD25-, Gr-1-, CXCR4-, CD62L-, CD49e-, CD44-, CD90-, Flk-1-, EpCAM- (all Pharmingen), and c-Kit-PE (SBA, Birmingham, AL); CD4- and CD8-PerCp (Pharmingen). Biotinylated B7.1 (CD80), B7.2 (CD86; SBA), and 1B2 antibodies (provided by J. Nikolic-Zugic, Sloan-Kettering Institute) were detected with streptavidin-PerCp or streptavidin-APC (Jackson ImmunoResearch Laboratories, West Grove, PA), and nonviable cells were detected with propidium iodide (PI). PE-rat IgG2a, FITC-hamster IgG (Serotec, Oxford, UK), and PerCp- and APC-streptavidin were used as controls. FACS sorting was performed using BD FACSAria, a high-speed sorter (acquisition rates of up to 70,000 events/s) with fixed-alignment cuvette flow cell and up to three air-cooled lasers at 488-, 633-, and 407-nm wavelengths. Cells were sorted in a cold and sterile environment for high and low PE staining (Sca-1). Sca-1^bright and Sca-1^dim cells were collected into different cold glass FACS tubes, and after cells were centrifuged, they were transferred to culture medium and incubated at 37°C, 7% CO₂ on plastic plates.

Immunohistochemistry
Cryostat and paraffin sections, cells growing on plastic, and glass smears of renal Sca-1⁺ cell preparations were immunoperoxidase labeled and specified antibodies and secondary reagent system, DAKO Envision + System, HRP (DAKO, Glostrup, Denmark), were applied using DAB as chromogen. Nuclei were counterstained by hematoxylin. The following antibodies were applied: Polyclonal rabbit anti-cytokeratin (wide-spectrum screening), polyclonal rabbit anti-cow S-100, mouse monoclonal anti-neurofilament (NF), and mouse monoclonal anti–-smooth muscle actin (all from DAKO); rat monoclonal anti-F4/80 (Serotec); rat monoclonal anti-CD31 (Chemicon Hampshire, UK); rat monoclonal anti–Sca-1 (BD Pharmingen); mouse monoclonal anti-vimentin (Clone VIM-13.2) and rabbit anti-laminin (both from Sigma); rabbit anti–-galactosidase (Abcam, Cambridge, MA; 1:2000, overnight incubation); and mouse monoclonal anti-CD45 (clone 2B11 + PD7/26; Dako). For immunofluorescent labeling, acetone-fixed sections of adult mouse kidney (6 µm) were incubated with a mixture of polyclonal rabbit anti–von Willebrand factor, polyclonal rabbit anti-cytokeratin (Dako), and rat anti–Sca-1 (BD Pharmingen). Nuclei were counterstained with Hoechst33342. The following secondary antibodies were applied: Texas Red–conjugated donkey anti-mouse antibody and Cy2-conjugated goat anti-rabbit antibody (both from Jackson ImmunoResearch Laboratories). Paraformaldehyde-fixed cultures were labeled with mouse anti-human nestin (CRP, Cumberland, VA) and rabbit anti-mouse -tubulin III (CRP) followed by incubation with donkey anti-mouse or anti-rabbit Cy3-conjugated secondary antibodies (Jackson). For all immunohistochemical stainings, a negative control was run using the same technique but omitting the primary antibody while adding the labeled secondary antibody.

Cell Culture
Renal Sca-1⁺Lin^– cells were incubated at 37°C, 7% CO₂ on plastic plates in culture with DMEM 1.0 g/ml glucose + 10% FCS + 1% penicillin streptomycin (Biological Industries, BetHaemek, Israel). After 7 d in culture, an enriched population of Sca-1⁺Lin^– cells was plated. Cultures were maintained with medium that was exchanged every 2 to 3 d, and transfers were done when needed. For establishment of single-cell clones, isolated Sca-1⁺Lin^– cells were suspended in 10 ml of culture medium. Cells were counted and diluted to reach an average of 1 cell/200 µl culture medium and replated into 96-well culture plates (200 µl/well), as described previously (25).

Estimation of Multipotency
For enhancement of differentiation of freshly isolated and cloned renal Sca-1⁺Lin^– cells into several cells type, 10⁵ intact cells were plated. Osteogenic differentiation was induced in culture medium, 50 µg/ml ascorbic acid, 10 mM -glycerophosphate, and 10^–8 M dexamethasone. Cell differentiation was tested 3 wk later. For detection of osteocytes, alizarin red staining (Sigma) and nitro blue tetrazolium staining (DAKO) for alkaline phosphatase were used as described previously (25). For enhancement of adipocyte differentiation, culture medium was supplemented with 1-methyl-3-isobutylxanthine, 10^–9 M dexamethasone, 5 mM insulin, and 5 mM indomethacin (Stemcell Technologies, Vancouver, BC, Canada). For detection of oil droplets, cultures were stained in saturated Oil-Red O solution (Sigma) in 60% isopropanol (25).

Neural differentiation was induced by supplementing the culture medium with human basic fibroblast growth factor (FGF; 100 ng/ml) during the first week of culture followed by FGF-8b (10 ng/ml) and brain-derived neurotrophic factor (10 ng/ml) during the second and third weeks, respectively. Neural differentiation was assessed by specific morphology and expression of nestin and -tubulin III (see above).

Array Processing
All experiments were performed using Affymetrix Mouse 430A2.0 oligonucleotide arrays, as described at http://www.affymetrix.com/support/technical/datasheets/rat230_2_datasheet.pdfURL1. Total RNA from each sample pool was used to prepare biotinylated target RNA, with minor modifications from the manufacturers recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Details of quality control measures can be found at http://eng.sheba.co.il/genomics.

Array Analysis
The hybridization results were analyzed using MAS 5. Probe sets with signals lower than 20 were filtered out. The t test was applied as a probability measure (P < 0.05) of Sca-1⁺ and Sca-1^– discrimination samples with P < 0.05 further filtered using a signal average 1.5-fold change threshold. Probe sets that were up- or downregulated by at least 1.5-fold were classified into functional groups using Go Annotation tools (Dennis G Jr et al., DAVID: Database for Annotation, Visualization, and Integrated Discovery, http://apps1.niaid.nih.gov/David/upload.asp).

Assay for Inhibition of Immune Stimulation in the 2C TCR Transgenic Mouse Model
Spleen cells of 2C transgenic H-2^b mice, expressing the TCR- with specificity for H-2L^d mice, were harvested, and cell suspensions were treated with Tris-buffered ammonium chloride to remove RBC and brought to a concentration of 2 x 10⁶ cells/ml in RPMI 1640 complete tissue culture medium at 37°C in a 5% CO₂/air incubator. The cells (2 x 10⁶/ml) then were stimulated with irradiated (20 Gy) DBA/2 splenocytes (2 x 10⁶/ml) in the presence or the absence of 10, 5, 1, and 0.5% cells of specific (DBA/2 origin) and nonspecific (SJL origin) newly isolated and cultured renal Sca-1⁺ cells and BM-derived MSC. Cultures were incubated for 72 h in 24-well plates. The inhibition of proliferation of specific effector T cells was monitored by cytofluorimetric analysis, measuring the level of 2C transgenic cells, specifically stained by the 1B2 antibody, directed against the clonotypic anti–H-2L^d TCR.

Ischemia/Reperfusion Injury and Cell Transfer
Ischemia/reperfusion (I/R) injury was performed by occluding the left renal pedicle for 30 min (11). A volume of 50 µl of cell suspension that contained newly isolated Sca-1⁺ cells from ROSA26 was injected directly into the left kidney through the renal pelvis with a 32-G needle shortly after removal of the vascular clamp. The needle was advanced into the renal parenchyma, and the cell suspension was injected slowly. For controls, (1) mice were subjected to an identical protocol of I/R but received PBS or Sca-1^– cells instead of Sca-1⁺ cells or (2) received the same amounts of cells without bearing ischemic injury.

	Results

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Progenitor Cells in the Adult Mouse Kidney
Taking into account that Sca-1 is expressed on renal parenchymal cells, mostly distal tubules in the cortex and medullary cords (27), we aimed to isolate a "tubular-depleted" fraction of kidney cells initially using mild digestion, which predominantly affects loosely bound cells and not those that are integrated within tight junctions, followed by gentle mechanical mincing and sequential filtration through a 70- and 40-µ mesh to deplete large cells and undigested tubular segments and tissue fragments. RBC were depleted with the addition of ACK (see Materials and Methods). This cell preparation does not represent all of the cells that are present in the adult kidney. The cell suspensions were co-stained with both PE-labeled anti–Sca-1 and FITC-labeled anti-CD45 to determine cells of hematopoietic origin. For control, the same method was applied to mouse spleen. Typical results of dual-wavelength FACS are shown in Figure 1. As can be seen in Figure 1, A and B, a distinct population in both spleen and kidney can be observed on the forward (FSC) and sidescatter (SSC) plot ("lymphogate"). Analysis of CD45 expression in the spleen within this gate reveals a predominant CD45⁺ cell population of hematopoietic origin, with the highest proportion of Sca-1^–CD45⁺ cells (tissue leukocytes) and to a much lesser extent Sca-1⁺CD45⁺ cells (Figure 1C). In the kidney preparation, we also could observe a CD45^– fraction that expresses the Sca-1 marker (Sca-1⁺CD45^– cells, representing 0.5 to 1% of the total cell suspension; Figure 1D). The finding that spleen cells, prepared under identical conditions, were devoid of this CD45^– fraction indicated that it was not an artifact resulting from loss of this surface marker during preparation of cell suspensions. It I interesting that as the size of the gate increased and contained smaller cells (according to the FSC), the Sca-1⁺CD45^– cell fraction increased. Moreover, exclusively gating the smaller cells (R1') revealed a more predominant Sca-1⁺CD45^– cell population at the expense of the Sca-1^–CD45⁺ phenotype (Figure 1D). When preparing cells without ACK to include RBC and staining for the erythrocyte antigen Ter119, we could show that although the majority of cells within the R1' gate are Ter119⁺Sca-1^–, a population of Sca-1⁺Ter119^– cells still exists (data not shown). Thus, within the kidney lymphogate, the cell preparation can be separated into CD45⁺ and CD45^– cells. We hypothesized that the Sca-1⁺CD45^– fraction contained renal-derived progenitor cells.

View larger version (52K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Identification of stem cell antigen-1–positive (Sca-1+)CD45– cells in adult mouse kidney. (A and B) FACS analysis of adult spleen (A) and kidney (B) cells: Representative forward (FSC) and side (SSC) scatter plot. Boxed cells in R1 represent the lymphogate in spleen and kidney, whereas R1' represents cells with smaller size in the lymphogate. (C and D) Spleen and kidney cells were stained further with Sca-1 and CD45 antibodies to distinguish cells of hematopoietic origin. A Sca-1+CD45– cell fraction is observed in the kidney within R1 and R1' (D).

View larger version (84K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. In situ detection of nontubular renal Sca-1+ cells in papilla. Frozen sections of adult kidney were immunostained with anti-mouse Sca-1. Shown is positive staining in medullary cords (A) and small arteriole (B). Diffuse staining was observed in the renal papilla (C), whereas control staining of the papilla without primary antibody was negative (D). (E and F) Double immunofluorescence was performed to detect Sca-1 expression (red) and cytokeratin (green). Images were overlaid to demonstrate co-localization of Sca-1 and CK (yellow). (E) Renal papilla. (F) Outer part of the papilla; nontubular Sca-1 cells are indicated in red (G through J) Several high-magnification fields of the papilla showing nontubular Sca-1 cells (red, arrows) in close proximity and adjacent to tubular cells (yellow). Magnifications: x10 in A, C, and D; x100 in B and F through J; x40 in E.

View larger version (104K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. In situ detection of nontubular renal Sca-1+ cells in medulla and cortex. Double immunofluorescence was performed to detect Sca-1 expression (tubular and nontubular; green, A, D, and G) and von Willebrand factor (vWF), which stains endothelial cells (red; B, E, and H) in medullary sections. Overlaid images are shown in (C, F, and I). Lower magnification (A through C) and higher magnification (D through F) of the same field, as well higher magnification of a different field, all showing nontubular Sca-1 cells (arrows). Magnifications: x40 in A through C; x100 in D through F.

View larger version (58K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Isolation of nontubular renal Sca-1+ cells. Sca-1+ cells were enriched by the MACS system with anti–Sca-1 microbeads. (A) Representative FSC and SSC plot of the enriched cells, which is separated into three boxed areas (the lymphogate R1, as well as R2 and R3). (B) After sorting at least twice, approximately 90% of the cells expressed Sca-1, whereas the negative fraction contained <1% of Sca-1+ cells. Cells that were stained with Sca-1 and CD45 antibodies demonstrate approximately 10-fold enrichment in the R1 Sca-1+CD45– cell fraction. (C and D) The R1 through R3 fractions were analyzed further by cell sorting and immunostaining of sorted cells for Sca-1, CD45, vimentin, and pancytokeratin (CK). (C) R1 cells in the upper panels show differential expression of Sca-1 (intense staining in smaller cells [arrows] and patchy staining in larger cells [arrowheads]), whereas R1 cells in lower panels positively stain for vimentin and fail to stain CD45. (D) R2 sorted fraction is shown to be composed of cell fragments that are cells that are strongly positive for Sca-1, whereas tubular segments that express CK are found in R3. Magnification, x100.

View larger version (43K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Flow cytometric analysis of nontubular Sca-1+ cells. Enriched Sca-1+ cells were labeled with Sca-1 plus the indicated (A) hematopoietic lineage (upper row) and progenitor cell markers (lower row) and (B) mesenchymal (upper row), epithelial, and immunologic markers (lower row). Double staining by FACS shows the frequency of Sca-1 and the various CD markers. In all samples, cells are gated in R1. (C) R1 sorted cells were stained with Sca-1 antibody and further sorted into Sca-1bright and Sca-1dim cells. (D) Isotype and streptavidin control staining.

Culture and Cloning of Renal Sca-1⁺Lin^– Cells
An enriched nontubular Sca-1⁺ cell fraction was isolated from adult murine kidneys by magnetic beads and cultured with enriched medium that contained FCS (see Materials and Methods). At culturing, renal Sca-1⁺ cells are floating cells, whereas within 24 to 72 h, a fraction of cells adhere to the culture dish and the remaining nonattached fraction consists of contaminating tubular segments, floating cell aggregates, and round cells (Figure 6A). Thus, renal tubular segments are discarded after short-term culture by continuous removal of the nonadherent fraction. Within 7 d, the Sca-1 culture consists of an adherent population of small round cells, whereas spindle-like and elongated shapes start to appear and increase with time (Figure 6, B and C). Flow cytometric analysis of trypsinized adherent cells 14 d in culture revealed decreased Sca-1 levels compared with fresh Sca-1⁺ cells; nevertheless, the former maintained a stable Sca-1⁺Lin^–CD45^– phenotype (of the Lin analysis, only CD11b is shown). Moreover, cells typically failed to stain for c-Kit and CD44 (Figure 6D). Immunostaining on culture dishes revealed positive Sca-1 staining and weak immunoreactivity to -smooth muscle actin (Figure 6, E and F, respectively), whereas pan-cytokeratin, CD31, NF, and S-100 were negative (data not shown).

View larger version (72K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Cultured renal Sca-1+Lin– cells. (A through E) Culture of enriched nontubular Sca-1+ cell fraction after MACS. (A) Nonadherent fraction showing cell aggregates that positively immunostained for CK. (B and C) Phase contrast images of adherent fraction at 7 d in low (B) and higher (C) magnification, showing the appearance of spindle-like and elongated cells. (D) Flow cytometric analysis of trypsinized cells after 14 d in culture. Double staining by FACS shows the frequency of Sca-1 and the surface markers CD45, CD29, CD11b, CD44, and c-Kit. Immunostaining of elongated cells for Sca-1 (E) and -smooth muscle actin (F). (G through I) Culture of FACS-sorted subpopulations. Phase contrast images of adherent nontubular R1 Sca-1bright cells at 24 h (G) and 28 d (H) and nonadherent nontubular R1 Sca-1dim cells, which remain round cells and are not viable (I). (J through L) Double immunofluorescence of cultured R1 Sca-1bright cells for vimentin (red; J), cytokeratin (green; K), and overlaid image with nuclear counterstaining (Hoechst 33342; L). Magnifications: x40 in A, E, and F; x100 in J through L.

To determine further the pluripotency of nontubular Sca-1⁺Lin^– cells, we tested their capacity to differentiate into various types of cells after induction. For elimination of the possibility that the Sca-1⁺ cell population contains several progenitors that give rise to the different cell types, it is important to perform this analysis at the single-cell level. We therefore established clones of renal Sca-1⁺Lin^– cells using standard single-cell cloning methods. In some experiments, after 10 d in culture to let cells adhere and elimination of the nonadherent fraction, single small round cells were isolated by limiting dilution into 96-well plates. Initially, these cells were quiescent, but they slowly started to proliferate, so by 4 wk, small clones of dispersed cells were formed, whereas by 8 wk, we observed cell aggregates that resembled neurospheres, previously identified in cultures of neuronal and skin stem cells (25) (Figure 7, A and B). After 10 wk, the clones were dissociated and moved to 24-well plates and then into 12-well plates over a period of 3 wk and subsequently into small flasks. Three independent clones of renal Sca-1⁺ cells were established. To date, the clonal isolates have been grown in culture for more than 12 passages with a doubling time of approximately 36 h. Immunocytochemical analysis of a representative cell clone revealed uniform expression of vimentin and laminin but not cytokeratin (Figure 7, C and D), whereas some cells were detectably positive for -smooth muscle actin and others showed a migratory appearance when nonconfluent (Figure 7, E and F). It is interesting that we found heterogeneous cell populations expressing CD31, a marker of endothelial cells (Figure 7, G and H). We then examined whether these cells could differentiate into osteocytes, adipocytes, and cells of neuronal lineage (Figure 8). When cloned cells were cultured with MDI-I mixture for 21 d, most of the cells (95% per plate) showed cytoplasmic accumulation of oil droplets stained with Oil-Red O, indicating that Sca-1⁺ cells differentiated into adipocytes (Figure 8, B and B1). When treated with osteogenic inducers, 21 d after induction, a large population of cells (95% per plate) showed the presence of alkaline phosphatase, one of the early markers of osteogenesis, and stained positive with calcium-binding alizarin red, both indicating osteogenic differentiation (Figure 8, D and F). After induction, neuronal lineage differentiation of renal Sca-1⁺Lin^– cells was confirmed by staining with nestin and -tubulin III. Although neuronal-like projections were visible in cultures, >90% of the cells stained positively for both nestin (Figure 8H) and -tubulin III (Figure 8J), confirming the neuronal identity of these cells. In all instances, control medium failed to induce adipogenic, osteogenic, and neuronal differentiation (Figure 8, A, C, E, G, and I, respectively). Under these same conditions, BM-derived MSC differentiated into the mesodermal cell types at comparable frequencies (data not shown), whereas control 3T3 mature fibroblasts could be differentiated into adipocytes but not to any other cell type. Thus, the renal Sca-1⁺Lin^– cells are multipotent and demonstrate remarkable plasticity.

View larger version (108K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Characterization of cloned renal Sca-1+Lin– cells. Clonal-derived cells demonstrate the formation of spheres (A, arrows, and B). Immunostaining of cloned cells shows positive staining for vimentin (C) and lack of staining for CK (D). Some cells were detectably positive for -smooth muscle actin (E) and CD31 (G and H). Under nonconfluent conditions, a migratory dendritic-like phenotype can be observed (hematoxylin staining). Magnifications: x10 in A, C, D, and G; x40 in B; x100 in E and H.

View larger version (79K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Clonal derivation of adipogenic, osteogenic, and neuronal lineages. Highly efficient differentiation of cloned renal Sca-1+Lin– cells (90 to 95% of plated cells) into adipogenic (B and inset B1), osteogenic (D and F), and neuronal lineages (H and J) is demonstrated. Cell differentiation in these cultures of cloned cells was observed after staining with oil red O (A and B, red-orange), nitro blue tetrazolium (C and D, blue), and alizarin red (E and F, red) and by immunostaining with an antibody specific for nestin (G and H, red) and for -tubulin III (I and J, red). Magnification, x10.

Microarray profiling (Table 2) was concordant with the FACS analysis of cell surface markers, specifically showing genes encoding for lymphocyte antigen 6 complex, locus A (Sca-1), and integrin-1 (CD29) to be significantly upregulated in the Sca-1⁺ fraction, and adding the tetraspanin (CD151) and transferrin receptor (CD71) as surface markers that are present. Sca-1⁺ cells were enriched for multiple transcriptional repressors and cellular quiescence genes (Hipk2, Max1, Nf2, Ndr2, Sav1, quiescin Q6, and klotho [33–39]) as well as genes that participate in resistance to stress, with upregulated detoxifier systems and antioxidative genes (Table 2). These along with TGF-, Yes (Yamaguchi sarcoma), and gp130 (IL6st) signaling and the interaction with the extracellular matrix via integrin-1 and Adam9 (all upregulated in the Sca-1⁺ cells) are essential attributes of the recently described core stem cell properties (or "stemness") (40). It is interesting that renal Sca-1⁺ cells overexpress developmental pathways that are associated with mesoderm lineage formation. These include members of the TGF-/bone morphogenic protein (BMP) family, which prompt efficient myogenesis and osteogenesis (41); furin, a TGF-1 activator; TGF- receptor II; and Sitpec/Ecsit, which associates with Smad1 and Smad4 and is required for BMP signaling and mesoderm formation during mouse embryogenesis (42,43). Accordingly, a variety of muscle and bone genes that are implicated in these tissues’ formation are upregulated in renal Sca-1⁺ cells: Myocyte enhancer factor 2A and YY1-associated factor 2 (YAF2) (myocardiogenic transcription factors that have been shown to be critically involved in the development and phenotype regulation of skeletal and cardiac muscle [44–46]); Filamin- (filamentous actin cross-linking protein, which accelerates differentiation into myotubes [47]); calumenin and fractured callus expressed, which are elevated in the early stages of a healing fracture and during differentiation along the osteoblast lineage; and exotoses (multiple)2, implicated in heparan sulfate proteoglycans synthesis and bone overgrowth (48–50). In addition, 6MPR/IGFIIR, insulin-induced gene 2, and Prss11 (IGF-binding protein protease), which promote adipogenic differentiation, are overexpressed in renal Sca-1⁺ cells (51,52). Other developmental signaling pathways might be active in renal Sca-1⁺ cells on the basis of the expression of specific genes, such as LRP6, a critical mediator of Wnt signaling in mice (53), and FGFR1 (54). Finally, overexpression of a variety of genes that are involved in cell adhesion, motility, and cytoskeleton assembly (e.g., Lasp1, Arpc3, Arpc5, nonmuscle Myosin IC) suggests a phenotype of migrating cells, a feature that is required if renal Sca-1⁺ cells were to participate in tissue repair.

View larger version (23K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Immune regulatory effects of renal Sca-1+Lin– cells. The inhibitory effect of renal Sca-1+Lin– cells was studied by one-way MLR in the 2C transgenic animal model. Splenocytes from 2C transgenic mice (H-2b) bearing transgene T cell receptor (TCR) specific against H-2d (1B2+) were stimulated in MLR against DBA/2 splenocytes (H-2d). FACS analysis shows the frequency of 1B2+ CD8 T cells in the absence (2C control, no inhibition; ) or the presence of newly isolated (left) and 30-d cultured (right) renal Sca-1+Lin– cells of DBA/2 origin () or SJL origin (dark gray bars). The effects of whole bone marrow (BM) cells (left) and mesenchymal stem cells (right) of DBA/2 origin () were analyzed for comparison. Data are shown 3 d after initiation of the mixed lymphocyte reaction.

View larger version (136K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. Contribution of renal Sca-1+Lin– progenitor cells to kidney repair. Cells were obtained from ROSA26 transgenic mice and injected directly into kidneys of C57BL/6 wild-type mice. (A and B) Immunostaining for LacZ was performed in control ROSA26 transgenic kidneys cells; note uniform positive staining. (C through F) Sections of kidney from C57BL/6 wild-type mouse 4 wk after ischemia/reperfusion (I/R) injury and transplantation of renal Sca-1+Lin– cells shown in low magnification (C and D) and high magnification (E and F); note positive staining in individual tubular cells (E) and interstitial cells (F, arrowheads). (G and H) Control C57BL/6 kidney subjected to I/R injury and injection of renal Sca-1– cells; note lack of staining of LacZ in corresponding deep regions. Magnifications: x10 in A, C, and G; x40 in B, D, and H; x100 in E and F.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our results using FACS to analyze renal cell suspension from adult mice showed a highly reproducible population termed "lymphogate" on the basis of size and granularity. When a sample of spleen was subjected to the same staining and run through the cell sorter immediately before a kidney sample, the renal lymphogate was found to be in the same gated region as the spleen lymphogate. In contrast with the spleen cells, further FACS analysis of the kidney lymphogate separated these cells into hematopoietic and nonhematopoietic fractions on the basis of the presence or absence, respectively, of the pan-leukocyte cell surface marker CD45. The CD45^– fraction was shown to express Sca-1 and was hypothesized to include the renal stem cells. This cell fraction is greatly enriched by Sca-1 magnetic cell sorting. Because Sca-1 has been reported to present in renal epithelial tubular cells, it was important to assess by additional FACS sorting whether the gated fraction included epithelial cells. The mesenchymal nature of the gated CD45^–Sca-1⁺ cell fraction was confirmed after sorting and phenotypical characterization that showed these cells to be positive for vimentin and negative for cytokeratin. Furthermore, morphologic examination showed that this cell fraction is not homogenous, containing at least two subpopulations that could be sorted further on the basis of Sca-1 expression into Sca-1^dim and less frequent Sca-1^bright cells. The latter were shown to be a population of highly adherent small round cells, which transform into fibroblast-like cells in culture. A similar cell phenotype also can be obtained after culture of the enriched nontubular Sca-1 population that was obtained by magnetic sorting and elimination of the nonadherent fraction. Therefore, it seems that additional FACS sorting of the kidney preparations according to size and granularity yields a fraction that includes both the putative stem cells and significant numbers of non–stem cells, and sorting for Sca-1^bright cells within the lymphogate will be advantageous. Clearly, the ability to isolate these cells constitutes a critical step in being able to observe, characterize, and understand them in future studies.

Whereas significantly expressing CD29 (1-integrin), the nontubular Sca-1⁺ cells showed little to no expression of several markers that are known to be present on other stem cells. For example, CD44, CD90, CD34, and c-kit, which are known to be enriched in BM mesenchymal and hematopoietic progenitor cells, were not expressed in the gated renal Sca-1⁺ cells (2,25). Therefore, we conclude that these CD45^–Lin^–Sca-1⁺ cells are different phenotypically from stem cells of BM but similar to those observed in other tissues, in particular, multipotential myogenic and cardiogenic progenitor cells in the interstitium of skeletal muscle and myocardium that are Sca-1⁺ but CD45^–, CD34^–, and c-Kit^– (20,21,58). We have identified Sca-1⁺ cells in the kidney interstitium in close proximity to tubules, most notably in the renal papilla. During preparation of this article, putative adult kidney stem cells were isolated with the aid of side population FACS sorting (59). It is interesting that the kidney side population cells, which were shown to promote kidney regeneration, reside in the interstitial space and are Sca-1⁺ (>70% on average) but CD45^–. In addition, populations of renal papillary cells were suggested as stem cells on the basis of the their ability to retain bromodeoxyuridine (label-retaining or low-cycling cells) (60). These cells, which were shown to be located mostly in the interstitium of the renal papilla, were phenotypically negative for CD45, CD34, CD44, or c-Kit (Sca-1 was not evaluated). Therefore, shared phenotypic features suggest that the nontubular Sca-1⁺Lin^– cells may resemble not only resident cardiac and skeletal stem cells but also several proposed renal progenitor populations.

Recently, Verfaillie and co-workers (61), who previously identified multipotent adult progenitor cells that co-purify with MSC in postnatal BM, extended their findings into solid organs. Accordingly, cells that were cultured from muscle and brain (negative for CD44, CD45, major MHC classes I and II, and c-kit) differentiated to mesodermal, neuronal, and endodermal cell types (62). These cells, which originally were derived from BM, might circulate and home to stem cell niches. In fact, Dreyfus et al. (63) recently showed that multipotent CD45^–Sca-1–expressing cells that are located in muscle connective tissue can derive from BM in adulthood, constituting a reservoir of interstitial stem cells. It is interesting that many known genes that are overexpressed in the renal Sca-1⁺ microarrays belong to response pathways to developmental signaling molecules, such as Wnt, TGF-/BMP, and FGF. These pathways, critical for self-renewal of stem cells (64), have been shown to be overexpressed in mesoderm progenitor cells of fetal origin, termed mesoangioblasts, in which they activate differentiation programs. Future studies using the formation of BM chimeras and lineage-tracking analysis can establish lineage relationships of the nontubular renal Sca-1⁺ progenitors and determine whether they are intrinsic to the organ (i.e., reside and self-renew in the kidney from early embryonic stages) or rather migrate at other developmental stages.

Recent studies have suggested that a significant part of the replenishment of the tubular epithelium after ischemia derives from intrinsic tubular cell proliferation rather than BM-derived cells (56,65). Indeed, transplantation of human HSC in ischemic kidneys suggests a role in vasculogenesis but not tubulogenesis (66). Because large numbers of proliferating cells are observed early after ischemic injury at diverse sites in the regenerating tubules, Duffield et al. (56) challenged the presence of endogenous kidney stem cells by arguing that a large population of such cells would have to be adjacent to tubules to give to their rise. Indeed, after ischemic injury and intrarenal injection of an enriched fraction of nontubular Sca-1⁺ cells, we observed localized rather than diffuse engraftment in tubular cells; therefore, contribution to tubular repair probably is minor compared with intrinsic tubular cell proliferation. Isolation, expansion, and delivery of the Sca-1^high cells into injured kidneys might provide improved results. In any case, the beneficial effects of nontubular Sca-1⁺Lin^– cells might be mediated by their ability to supply large amounts of angiogenic, antiapoptotic, and mitogenic factors in kidney disease (67). Furthermore, the multipotency of the cells and their immune regulatory potential call for their evaluation in both autologous and allogeneic models of injury that do not afflict the kidneys (e.g., heart infarct, neurodegenerative disease).

	Acknowledgments

 
This study was supported in part by grants from Tissera, Inc., Mrs. E. Drake, and the Gabriella Rich Center for Transplantation Biology Research.

Y.R. holds the Henry H. Drake Professorial Chair in Immunology. He serves as the Chairman of the Advisory Board and holds equity and has patent arrangements with Tissera, Inc. B.D. holds equity and has patent arrangements with Tissera, Inc.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

B.D. and L.Z. contributed equally to this work.

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