Biomaterials 50 (2015) 56e66

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Nestinþ kidney resident mesenchymal stem cells for the treatment of acute kidney ischemia injury Mei Hua Jiang a, b, c, 1, Guilan Li b, 1, Junfeng Liu b, d, 1, Longshan Liu e, Bingyuan Wu f, Weijun Huang b, Wen He g, Chunhua Deng h, Dong Wang i, Chunling Li j, Bruce T. Lahn b, Chenggang Shi k, **, Andy Peng Xiang a, b, l, * a Cell-gene Therapy Translational Medicine Research Center, The Third Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China b Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-Sen University, Guangzhou, China c Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China d Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China e Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China f Department of Cardiology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China g Department of Geriatrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China h Department of Urology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China i Department of Laboratory Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China j Institute of Hypertension & Kidney Research, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China k Department of Nephrology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China l Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 November 2014 Accepted 20 January 2015 Available online

Renal resident mesenchymal stem cells (MSCs) are important regulators of kidney homeostasis, repair or regeneration. However, natural distribution and the starting population properties of these cells remain elusive because of the lack of specific markers. Here, we identified post-natal kidney derived Nestinþ cells that fulfilled all of the criteria as a mesenchymal stem cell. These isolated Nestinþ cells expressed the typical cell-surface marker of MSC, including Sca-1, CD44, CD106, NG2 and PDGFR-a. They were capable of self-renewal, possessed high clonogenic potential and extensive proliferation for more than 30 passages. Under appropriate differentiation conditions, these cells could differentiate into adipocytes, osteocytes, chondrocytes and podocytes. After intravenous injection into acute kidney injury mice, Nestinþ cells contributed to functional improvement by significantly decreasing the peak level of serum creatinine and BUN, and reducing the damaged cell apoptosis. Furthermore, conditioned medium from Nestinþ cells could protect against ischemic acute renal failure partially through paracrine factor VEGF. Taken together, our findings indicate that renal resident Nestinþ MSCs can be derived, propagated, differentiated, and repair the acute kidney injury, which may shed new light on understanding MSCs biology and developing cell replacement therapies for kidney disease. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Nestin MSCs Acute kidney injury Paracrine VEGF

1. Introduction * Corresponding author. Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-Sen University, Guangzhou, China. Tel.: þ86 20 87335822; fax: þ86 20 87335858. ** Corresponding author. Tel.: þ86 20 82179168. E-mail addresses: [email protected] (C. Shi), [email protected] (A.P. Xiang). 1 These authors made equal contributions to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.01.029 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

Mesenchymal stromal/stem cells (MSCs) are multipotent progenitor cells that are able to differentiate into osteoblasts, adipocytes, and chondrocytes [1]. In addition to their self-renewal capacity and multipotency, MSCs also possess potent immunemodulatory and paracrine characteristics [2,3]. Furthermore, plenty of preclinical and clinical studies have identified the therapeutic potential of MSCs on kidney protection and repair by their

M.H. Jiang et al. / Biomaterials 50 (2015) 56e66

paracrine, anti-inflammatory, and immunomodulatory properties, such as acute kidney injury (AKI), chronic kidney disease (CKD) or renal transplantation [4e6]. Despites the major reservoir of MSCs in bone marrow, it has been identified that MSCs reside in almost all organs and tissues, these cells in association with the perivascular niche of these organs [7]. Although the renal resident MSCs have been isolated and shown phenotypic and functional equivalence with bone marrow MSCs, they also demonstrate distinct gene and protein expression profiles [8]. Because tissue resident MSCs might play an important regulatory role of tissue repair or regeneration, the unique functional roles of renal MSCs imply they might be more appropriate as cellular therapeutic agents for the treatment of kidney diseases [9,10]. However, our current understanding of tissue resident kidney MSCs remains limited. Conventionally, kidney MSCs are functionally isolated from renal tissue based on their capacity to adhere to the surface of culture flasks, but the isolated cells do not provide biological information about the starting population [11,12]. Because of the lack of the specific markers for kidney MSCs, the exact nature and localization of MSCs in vivo remain poorly understood. To overcome the limitations of these methods, there is a clear need for specific markers and methods to identify and prospectively isolate kidney MSCs. The intermediate filament protein, Nestin, is a widely employed marker of multi-potent neural stem/progenitor cells (NSCs) [13]. More importantly, Nestin labels adult stem/progenitor cell populations, indicating that Nestin might be a common marker of
ndez-Ferrer et al. firstly reported a multi-lineage stem cells [14]. Me stromal Nestin expressing population in bone marrow showing the typical characteristics of MSCs, suggesting Nestin might also become a specific marker for isolating the tissue resident MSCs [15]. Previous studies have shown the Nestin expression in adult and developing kidney [16], including repopulating mesangial cells [17], podocytes [18]. Under specific damage conditions, upregulation of Nestin expression was found in tubular cells, podocytes and interstitial cells [19,20]. To investigate whether Nestin can be used as a candidate marker for identifying the kidney resident MSCs, we report a method for isolating MSCs from the murine kidney, using flow cytometry in combination with in vitro function assays. Moreover, we study the reparative capability and the cellular and molecular mechanism of Nestinþ MSCs in acute kidney ischemia injury model. 2. Materials and methods 2.1. Mice Homozygous transgenic mice that expressed enhanced GFP under the control of a Nestin promoter (Nestin-GFP, on the C57BL/6 genetic background) were kindly provided by Dr. Masahiro Yamaguchi [21]. C57BL/6 mice were provided by Vital River Laboratories (Beijing, China). All animal studies were carried out in accordance with the guidelines of the Sun Yat-sen University Institutional Animal Care and Use Committee. 2.2. Isolation and culture of Nestinþ cells from the mice kidney Neonatal Nestin-GFP or C57BL/6 mice kidneys were harvested, the collected kidneys were incubated with Collagenase IV (300 U/ml; Sigma Aldrich, USA) and DNase I (100 U/ml; Sigma Aldrich, USA) in HBSS for 20 min at 37  C in a shaking water bath. Subsequently, the cell suspensions were digested with 0.25% trypsin (Sigma Aldrich, USA) at 37  C for 5 min and passed through a cell strainer with a mesh diameter of 40 mm, yielding single cells. The cells expressing green fluorescent protein (GFP) were sorted using an Influx Cell Sorter (BD, USA) and cultured on plastic plates in DMEM/F12 medium (Invitrogen, USA) with 20 ng/ml EGF (Peprotech, USA), 10 ng/ml bFGF (Peprotech, USA), 2% B27 (Invitrogen, USA), 1% N2 (Invitrogen, USA), and 100 IU/ml penicillin/streptomycin (Invitrogen, USA). The cells were cultured at 37  C under 5% CO2 and propagated every 2e3 days. 2.3. Colony forming unit-fibroblast assay For colony forming unit-fibroblast assay (CFU-F), 5000 Nestinþ cells were plated on tissue culture plastic and cultured in above-mentioned cell growth medium.

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After 10 days of cultivation, the number of colonies containing greater than 50 cells were assessed by inverted microscopy using an Olympus IX71 and quantified with the aid of a DP manager program (Olympus, Japan) [22]. 2.4. Cell proliferation assay Nestinþ cells proliferation was evaluated with Click-iT® EdU cell Fluor Cell Proliferation Assay Kit (Invitrogen, USA). The cells were labeled with 10 mM EdU for 24 h and then washed carefully to remove the dye and staining according to the manufacturer's instructions. Nuclei were counterstained with DAPI (Sigma Aldrich, USA). Cells were seeded into 12-well plates at a density of 10,000 cells/well. The cells were trypsin with 0.25% trypsin and counted for consecutive 6 days (n ¼ 3). Population doubling time (PDT) was determined with the following formula: PDT ¼ days in exponential phase/(log N2  log N1)/log 2 where N1 was the number of cells at the beginning of the exponential growing phase and N2 was the number of cells at the end of the exponential growing phase [23]. 2.5. Flow cytometry analysis Flowcytometry analysis and sorting were performed on FACS Calibur flow cytometer (BD, USA). The following anti-mouse antibodies were used: Sca-1-APC, CD44-PE, CD106-AF647, CD45-PE, CD11b-PE (eBioscience, USA). A minimum of 100,000 cells was acquired for each analysis. 2.6. Immunofluorescence staining analysis For immunofluorescence, the cells and kidneys were fixed in 4% PFA and dehydrated in 30% sucrose. The dehydrated kidneys were cut into 10 mm sections. The cells and sections blocked with 10% normal serum for 40 min, and then incubated with primary antibodies overnight at 4  C in a humidified chamber followed by incubation with then incubated with 488 or 594-conjugated secondary antibodies (Invitrogen, USA) at room temperature for 1 h. The following antibodies were used: anti-Nestin (1:200, Millipore, USA); anti-vimentin (1:500, Abcam, UK); antiPax2 (1:100, Abcam, UK); anti-NG2 (1:200, Abcam, UK); anti-PDGFR-a (1:200, Abcam, UK); anti-CD31 (1:200, BD Biosciences, USA); anti-podocin (1:400, Abcam, UK); anti-synaptopodin (1:300, Abcam, UK); anti-E-cadherin (1:200, Abcam, UK). All images were obtained using a Zeiss LSM710 confocal microscope. 2.7. Nestinþ cells differentiation ability in vitro For osteogenic differentiation, the cells were plated in 24-well plates at a density of 1  104 cells/well and cultured in the presence of ɑ-MEM (Invitrogen, USA), 20% FBS, 100 mg/ml ascorbic acid (Sigma Aldrich, USA), 100 nM dexamethasone (Sigma Aldrich, USA), 10 mM b-glycerophosphate (Sigma Aldrich, USA), and 100 IU/ml penicillin/streptomycin. The cells were fed every third day and maintained in culture for 3 weeks. Mineral deposition was visualized by Alizarin Red (Sigma Aldrich, USA) staining for calcium. For adipogenic differentiation, cells were induced in a DMEM-high glucose medium (Invitrogen, USA) with 100 nM dexamethasone (Sigma Aldrich, USA), 10 mg/ ml insulin (Sigma Aldrich, USA), 0.2 mM indomethacin (Sigma Aldrich, USA), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma Aldrich, USA), 10% FBS, and 100 IU/ml penicillin/streptomycin and maintained in culture for 3 weeks. Adipogenic differentiation was analyzed by Oil-Red O (Sigma Aldrich, USA) staining. For chondrogenic differentiation, cells were cultured in media containing serum free DMEM-high glucose, insulin-transferrinselenious (ITS) acid mix (BD Biosciences, USA), 50 mg/ml L-ascorbic acid 2-phosphate (Sigma Aldrich, USA), 1 mM sodium pyruvate, 0.1 mM dexamethasone (Sigma Aldrich, USA), and 10 ng/ml transforming growth factor b1 (TGFb1) (Cell Signaling Technology, USA). Medium was changed every 4 days for 3 weeks and then fixed in 4% paraformaldehyde for 15 min, monolayer cells were stained for sulfated proteoglycans with 1% Alcian Blue. For podocytic differentiation, cells were passed onto dishes that coated with type I collagen (0.1 mg/ml) and incubated in RPMI1640 media containing 5% FBS, 100 IU/ml penicillin/streptomycin, 2 mM glutamine and 10 mM all-trans RA for one week. After immunofluorescence staining for podocin and synaptopodin, the cells were examined via Zeiss LSM 710 confocal microscope (Zeiss, German). 2.8. Reverse transcription and quantitative PCR The total RNA was extracted from kidney tissue and cells using RNeasy Mini Kits according to the manufacturer's instructions (Qiagen, USA). One microgram of RNA was reverse transcribed using the RevertAid First Strand cDNA kit (Fermentas, USA). Quantitative PCR were performed as described elsewhere [24]. The primer details are shown in Table 1. 2.9. Acute kidney ischemia-reperfusion injury and cells transplantation To evaluate the recovery of renal function of Nestinþ cells in vivo, Nestinþ cells were washed with PBS and stained with the red fluorescent dye CM-DiI according to the manufacturer's instructions. Seventy-eight C57BL/6 mice (n ¼ 5 for each time point in control mice; n ¼ 8 for each time point in cell or conditioned media

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Table 1 Primers for Q-PCR. Genes

Forward sequence

Reverse sequence

Product size

Nestin Collagen II Collagen X SPARC Runx2 FabP4 PPARg Podocin Synaptopodin VEGF IGF-1 HGF G-CSF FGF2 GAPDH

50 -TCGCCAGGGAGGAGGCCATT-30 50 -AGTACCTTGAGACAGCACGAC-30 50 -CAGCAGCATTACGACCCAAG-30 50 -TTGGCGAGTTTGAGAAGGTATG-30 50 -CGTGGCCTTCAAGGTTGTA-30 50 -AATCACCGCAGACGACA-30 50 -CTGACCCAATGGTTGCT-30 50 -GTGTCCAAAGCCATCCAGTT-30 50 -ATGCTGCTTTCTCTATCCCC-30 50 -AGGAAAGGGAAAGGGTCAAA-30 50 -CCTCTTCTACCTGGCGCTCT-30 50 -CAAACTTCTGCCGGTCCTGT-30 50 -GTCTGGAGAGCTGTGGACACA-30 50 -CCCCAAGCGGCTCTACTG-30 50 -ACCACAGTCCATGCCATCAC-30

50 -CTCCCCAGCCCTCCCCAGAC-30 50 -AGTCTCCGCTCTTCCACTCG-30 50 -CCTGAGAAGGACGAGTGGAC-30 50 -GGGAATTCAGTCAGCTCGGA-30 50 -GCCCACAAATCTCAGATCGT-30 50 -GTGGAAGTCACGCCTTTC-30 50 -CAGACTCGGCACTCAATG-30 50 -GGCAACCTTTACATCTTGGG-30 50 -CCCTAGACTGCCTTCTCGT-30 50 -CGCCTTGGCTTGTCACAT-30 50 -GGGCTGCTTTTGTAGGCTTC-30 50 -GAAGGCCTTGCAAGTGAACGTA-30 50 -TGTGTCCACAGCTCTCCAGAC-30 50 -CAACTGGAGTATTTCCGTGACC-30 50 -TCCACCACCCTGTTGCTGTA-30

194bp 157bp 287bp 177bp 156bp 158bp 168bp 117bp 185bp 192bp 243bp 228bp 301bp 263bp 150bp

Glomerulus

A

Interstitial

B

Nestin-GFP

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1d

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90d

1 0.8 0.6 0.4 0.2 0

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90d

Fig. 1. Nestin expression in the glomeruli and interstitial of the Nestin-GFP mouse kidney. (A) Distribution of Nestin-GFP-positive cells in the mouse glomeruli at postnatal day 1, 7, 30 and 90. Scale bars: 50 mm. (B) Distribution of Nestin-GFP-positive cells in the mouse interstitial at postnatal day 1, 7, 30 and 90. Scale bars: 50 mm. (C) Quantitative real-time PCR analysis showed that the level of Nestin expression in the kidney decreases with age. The Nestin mRNA levels were normalized to GAPDH. Data are shown as the mean ± SEM.

M.H. Jiang et al. / Biomaterials 50 (2015) 56e66 treatment mice) were anesthetized with ketamine (150 mg/g) and xylazine (30 mg/g). Ischemia was induced by clamping the renal pedicles with non-traumatic microaneurysm clamps (Roboz Surgical Instruments, USA), which were removed after 25 min. One day after ischemic injury, 200 ml of a cell suspension containing 2  106 CM-DiI labeled or unlabeled (for the apoptosis assay) Nestinþ cells were slowly injected into the tail vein. Vehicle-treated control mice received 200 ml of PBS. Renal function was determined by the measurement of serum creatinine and BUN levels using Crea-plus kits (Roche Diagnostics, CA). At 0, 2, 4, or 7 days after surgery, the animals were sacrificed and serum samples and kidneys were collected. The number of integrated cells in the kidney was counted from 10 different fields at 400 magnification for each sample and averaged.

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random fields. The results are expressed as the percentages of apoptotic cell numbers in each section (n ¼ 3). 2.12. Conditioned medium transplantation in vivo Nestinþ cells at ~80% confluence were switched to serum-free DMEM/F12 and cultured for 48 h. Then, conditioned media (CM) were collected and concentrated 30 times using ultrafiltration membranes (molecular weight cut off: 3 kDa, Millipore, USA). About 200 ml control DMEM/F12 media or concentrated CM was injected into each renal injury mouse through tail vein. 2.13. RNA interference

2.10. Analysis of renal morphology

Vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) and negative transfection control siRNA (NTC) were purchased from Ribobio (Ribobio, China). Cells were transfected with double-stranded siRNA oligonucleotides specific for VEGF (sense sequence: 50 -GGCUUACCCUUCCUCAUCUdTdT-30 , anti-sense sequence: 50 -AGAUGAGGAAGGGUAAGCCdTdT-30 ). Nestinþ cells were incubated with 50 nM siRNA for 5 h and changed fresh DMEM/F12 media. Forty-eight hours after transfection, the CM were collected and used for the further experiments. The secretion of VEGF from Nestinþ cells was measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, USA). ELISA was performed according to the manufacturer's instructions. All samples and standards were measured in triplicate.

For analysis of mouse renal morphology, the collected kidneys were fixed in 4% paraformaldehyde at 4  C overnight and then embedded in paraffin. Sections of 5 mm thickness were stained with hematoxylin and eosin (H&E) and Masson reagents. In each experimental group, 10 high-power fields (HPF, 400) of renal section were randomly selected for assessing tubular injury and graded from 0 to 4, as outlined by Jablonski et al. [25,26]. All scoring was performed in a blinded manner. 2.11. Apoptosis assay Apoptotic nuclei were detected by using terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) staining kits (Roche Applied Science, USA) according to the manufacturer's instructions. The nuclei were counterstained with DAPI (Sigma Aldrich, USA). Images were collected using a Zeiss LSM710 confocal microscope. The percentage of TUNEL-positive cells was counted in nine

A

2.14. Statistical analysis All data are presented as the mean ± S.E.M obtained from at least three independent experiments. Comparison between groups was performed by the One-way

B

Bright field

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Primary culture

0.16%

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P0 Nestin -GFP

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C Nestin

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99.3 9.4%

30.1 2.9%

1.2 0.1%

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Counts

99.9 9.5%

0

0 100 101 102 103

Sca-1

0 100 101 102 103

CD44

100 101 102 103

CD106

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CD11b

Fig. 2. Isolation and culture of Nestinþ cells from 7 days old mice. (A) Flow cytometry analyses of renal cells from postnatal 7 days C57BL/6J (Control) and Nestin-GFP mice are shown as scatter plots. The cells in the oval represent GFPþ cells. (B) Schematic of the protocol used for Nestinþ cell culture; the primary cells generated was designated P0. Scale bar: 50 mm. (C) Multiplex immunofluorescence staining revealed that Nestinþ cells co-expressed Nestin (green), as well as vimentin (red), PDGFR-a (red), NG2 (red) and Pax2 (red), but not CD31 (red). Scale bars: 20 mm. (D) Flow cytometry histograms showed the expression of the indicated cell surface markers on Nes-GFPþ cells, including Sca-1, CD44, CD106, CD45 and CD11b.

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ANOVA with NewmaneKeuls post hoc comparison, with P < 0.05 considered statistically significant.

3. Results 3.1. The spatial and temporal expression profile of Nestin in the neonatal and adult kidney

The levels of mRNA encoding Nestin was also observed using quantitative real-time PCR in kidney tissues and shown in similar expression phenomenon with histological analysis (Fig. 1C). Thus, Nestin is continuous presence in neonatal and adult in kidney of mice. 3.2. Isolation and characterization of renal Nestinþ cells from the Nestin-GFP transgenic mice

It is has been shown that Nestin is expressed in developing and adult kidneys [16,18]. To further verify whether Nestin could be a potential marker for renal resident MSCs, we first evaluated the pattern of Nestin expression in the kidneys of Nestin-GFP transgenic mice at different times in the post-natal period via the microscopic examination of GFP synthesis. As a representative of Nestin expression, GFP fluorescence were observed in the kidney of post-natal 1, 7, 30, and 90 days mice, they were mainly distributed in glomeruli (Fig. 1A) and interstitial (Fig. 1B). In the adult mice, there were still a small number of resident GFPþ cells.

We isolated Nestinþ cells from the kidney of 7 day old NestinGFP mice through FACS sorting. There were approximately 1.11% GFPþ cells among the total cell population and almost all cells expressed GFP fluorescence (Fig. 2A, B). Immunofluorescent staining showed that Nestin co-expressed with vimentin, PDGFR-a, NG2 and Pax2. However, we did not find the endothelial marker CD31 expression (Fig. 2C). Because Nestin labels populations of stem/ progenitor cells, such as self-renewing MSCs in bone marrow [15,27]. We then analyzed some mesenchymal cell markers using

B Plate low density 1 x 5,000 50 000 00 00 /plate //pla pllat atte e 10days Dissociated Nestin+ cells

CFU-F counting

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C DAPI

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EdU positive cells (%)

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70 60 50 40 30 20 10 0

F Cell number (104)

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Fig. 3. Clony-forming and proliferation ability of Nestinþ cells. (A) Schematic of the experimental procedure used to generate single clones when the Nestinþ cells were cultured at a density of 5000 cells/plate. (B) CFU-F numbers were counted during passage 5, 15, and 25. (C) Representative images showed that proliferative Nestinþ cells were stained for EdU in passage 5, 15, and 25, respectively. Scale bars: 50 mm (D) Quantitative analysis showing the percentage of EdU-positive Nestinþ cells in each indicated passage. (E) Growth curves of the Nestinþ cells as assessed by direct counting. (F) Cytogenetic analysis showed that Nestinþ cells had a normal karyotype at passage 25.

M.H. Jiang et al. / Biomaterials 50 (2015) 56e66

flow cytometry and identified that Nestinþ cells were positive for Sca-1 (99.9% ± 9.5%), CD44 (99.3% ± 9.4%), CD106 (30.1% ± 2.9%) and negative for CD45 (1.2% ± 0.1%) and CD11b (1.0% ± 0.1%) (Fig. 2D).

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complement (Fig. 3F). However, at passage 50, 14% of the cells had hypodiploid or hyperdiploid chromosome numbers ranging from 38 to 60. For subsequent experiments, Nestinþ cells were used between passages 5 and 20. Taken together, these results indicated that Nestinþ cells could be considered as undifferentiated cell population and capable of proliferation in vitro culture.

3.3. Colony forming unit-fibroblast assay and proliferation ability of Nestinþ cells in vitro To determine whether renal Nestinþ cells contain stem cell-like cells, we studied the self-renewing ability by colony forming assay. Nestinþ cells were dissociated to single cell suspensions and plated at low density (5000 cells/in 10 cm2 plates) in expansion medium at passages 5, 15 and 25, respectively (Fig. 3A). After 10 days culture in vitro, colonies derived from single cells were calculated as CFU-F and the number of CFU-F formation was similar among different passages (Fig. 3B). We further compared the proliferation ability of Nestinþ cells through detecting the EdU incorporation at different passages and found the percentage of EdU-positive cells were similar at passages 5 (60.1% ± 3.1%), 15 (58.9% ± 2.8%) and 25 (58.6% ± 2.7%), respectively (Fig. 3C, D). In addition, these cells maintained stable population doublings and the PDTs of different passage cells were 45.1 h (P5), 46.8 h (P15) and 48.0 h (P25), respectively (Fig. 3E). Collectively, the capacity of clones forming and proliferation were similar in each passage. To examine karyotype, we cytogenetically analyzed Nestinþ cells at passages 25 and 50. At passage 25, all cells had a normal diploid chromosomal

3.4. The multi-potent differentiation capacity of Nestinþ cells To determine the extent of cell plasticity, Nestinþ cells were cultured under conditions that were favorable for osteogenic, adipogenic, chondrogenic and podocytic differentiation. The cells differentiated successfully and expressed tissue-specific markers, as shown by histochemical staining and Q-PCR (Supplemental Fig. 1). To further confirm the plasticity of the clonal (single cellderived) Nestinþ cells, we performed a multiple lineage differentiation assay with clones derived from a Nestinþ single cell, which plated into 96-well plates by limiting dilution (Fig. 4A, B). Subsequently, we picked two rapid proliferating clones and expanded them. After Nestinþ single clones growing in differentiation media, they all exhibited the capacity of multipotent differentiation. All two clones showed osteogenic and chondrogenic differentiation, assessed by Alizarin Red and Alcian Blue and expression of bone and cartilage-related genes, including SPARC, Runx2, CollagenⅡ and

B

A

Osteogenic Differentiation 1-3 weeks

Adipogenic Chondrogenic

Alizarin Red

Oil red O

Alcian Blue

Podocin

Synaptopodin

Alizarin Red

Oil red O

Alcian Blue

Podocin

Synaptopodin

Clone 2

C

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Podocytic

40 Relative mRNA expression

D

30

clone 1 control clone 1 differentiation clone 2 control clone 2 differentiation

20 10 0 A SP

RC

x2

n Ru

4 bP a F

A PP

R

II nX en g ge a a l l l l Co Co

in in oc od d p o Po pt na y S

Fig. 4. Clonal derived Nestinþ cells show the capacity of multi-potent differentiation in vitro. (A) Representative image showed the clone derived from single cell. (B) Schematic of the protocol used for multi-potent differentiation of clonal derived Nestinþ cells. (C) Single clones differentiated into osteocytes (alizarin red), adipocytes (oil red O), chondrocytes (alcian blue) and podocytes (podocin and synaptopodin). Scale bars: 50 mm. (D) Differentiated Nestinþ cells were examined by Q-PCR analysis for expression of osteogenic (SPARC, Runx2), adipogenic (FabP4, PPARg), chondrogenic (Collagen Ⅱ, Collagen Ⅹ) and podocytic (podocin, synaptopodin) markers. Expression levels of each gene were compared to undifferentiated Nestinþ cells (before differentiation; defined as 1). Data are shown as the mean ± SEM.

M.H. Jiang et al. / Biomaterials 50 (2015) 56e66

tubular necrosis, dilatation, swelling and fibrosis (grade 3.7). Compared with control mice, the animals injected with Nestinþ cells showed a marked alleviation of the renal injury, as indicated by the presence of fewer necrotic and dilated tubules and the preservation of cell structural integrity after the induction of ischemia (grade 2.1, P < 0.05). We further analyzed the level of protection from cell death by TUNEL staining. The numbers of apoptotic nuclei were quantified per total number of nuclei in each section. Interestingly, the number of TUNEL-positive cells increased at 4, 7 days after injury in control mice. In contrast, Nestinþ cell treatment obviously decreased the extent of apoptotic cell death (P < 0.05) (Fig. 5F), which indicated that Nestinþ cells might migrate to the injured areas of the kidney and could repair the injured kidney by differentiating and replacing the damaged cells, or improving the survival of the resident cells. As shown above, Nestinþ cells participate in renal injury repair. These cells might be capable of migrating toward and differentiating in the kidney. We traced the fate of injected Nestinþ cells using CM-DiI labeling, as Nestin expression is down regulated in most “differentiated” cells. Subsequently, both kidneys were

CollagenⅩ. The picked clones also underwent adipogenic differentiation, they accumulated lipid droplets that stained for Oil Red O, and exhibited upregulation of PPARg and FabP4. In podocytic medium, they began to express the podocyte markers podocin and synaptopodin at both protein and mRNA levels (Fig. 4C, D). 3.5. Treatment with Nestinþ cells improve renal function in mice with acute ischemic kidney injury To investigate whether injected Nestinþ cells assisted in kidney recovery after ischemia injury, renal function was assessed by the serial measurement of serum creatinine and BUN levels. The creatinine and BUN levels in PBS-injected mice were obviously increased after injury, peaking on day 4 and declining by day 7. Remarkably, the injected Nestinþ cells mice showed the significantly decreased serum creatinine and BUN levels at day 4 compared with control mice（P < 0.05）(Fig. 5A, B). The improvement of renal function was confirmed by the grading scores of renal tubular necrosis (Fig. 5C) and histological analysis (Fig. 5D, E). On day 4 after injury, the kidneys of control mice (injected with PBS) demonstrated notable damage, including renal

1.6

0.8 0.4

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*

100 50 0

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Fig. 5. Nestinþ cell transplantation improves renal function and inhibits structural deterioration after acute kidney ischemia injury. (A, B) Serial serum creatinine and BUN levels were measured in mice that received Nestinþ cells or PBS 1 day after ischemic injury. The treatment with Nestinþ cells significantly reduced the peak of serum creatinine and BUN levels 4 days after injury. (Data are expressed as the mean ± SEM; *P < 0.05). (C) Jablonski grading score was lower in Nestinþ cells treated mice than the control group (*P < 0.05). (D, E) Representative images showed H&E or Masson staining of renal tissue sections in each experimental group at 4 days after injury. Scale bars: 50 mm. (F) Nestinþ cells treatment reduced the extent of post-ischemic apoptosis in the kidney. Representative photograph showed TUNEL staining of renal tissue sections at 4 and 7 days after injury. TUNEL-positive cells were stained red, and the nuclei were shown in DAPI blue. Scale bars: 50 mm. Quantitative analysis showed the percentage of TUNEL-positive cells in each experimental group at 4 and 7 days after ischemic renal injury. (Data are expressed as the mean ± SEM, *P < 0.05).

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harvested from each mouse, and frozen sections were analyzed for the detection of the labeled cells. The CM-DiI labeled cells were colonized into renal interstitial (1.0% ± 0.06%), tubules (0.8% ± 0.04%) and glomerulus (0.6% ± 0.03%) at 400 magnification, respectively (Supplemental Fig. 2AeD). Furthermore, CM-DiI positive cells expressed the epithelium marker, E-cadherin in tubules and podocyte-specific markers, podocin and synaptopodin in glomerulus (Supplemental Fig. 2E, F), but not Nestin (data not shown). 3.6. Effects of Nestinþ cells CM on acute ischemic kidney injury in vivo Because Nestinþ cells did not integrate efficiently into kidney tubules, interstitial and glomeruli, these cells seem to be able to improve the survival of the resident renal cells by paracrine mechanism. Previous studies have demonstrated that paracrine factors may play the critical role in the therapeutic mechanisms of MSCs [28,29]. To investigate whether the Nestinþ cells participate to renal tissue repair through paracrine action, we injected the CM from Nestinþ cells to the acute ischemic kidney injury mouse model. Notably, 3 days after CM treatment, serum creatinine and BUN levels showed significantly decreased compared with the control group (P < 0.05) (Fig. 6A, B). Additionally, histological analysis showed that Nestinþ cells-CM treatment markedly ameliorated tissue damage, reduced necrosis and fibrosis by H&E and Masson staining, respectively (Fig. 6C, D). 3.7. VEGF released by Nestinþ cells participate in the renal injury repair To gain a deeper understanding of a potential renal-protective mechanism underlying the functional benefits observed from the treatment of Nestinþ cells CM, we screened the previously reported growth factors and cytokines with renal reparative function [30,31], including VEGF, insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), Granulocyte-colony stimulating factor (G-CSF), Fibroblast growth factors (FGF2) by Q-PCR analysis. We found that Nestinþ cells expressed high levels of VEGF (Fig. 7A). To

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4. Discussion In the present study, we demonstrate that the interstitial and glomeruli of kidney contain a population of Nestinþ MSC-like cells. We prospectively isolated the renal Nestinþ cells by FACS from Nestin-GFP transgenic mice. The Nestinþ cells exhibited properties of mesenchymal stem cells, including self-renewal capacity, clonogenicity and multipotency. We examined the impact of Nestinþ MSCs on the renal repair after acute ischemic injury. The delivered Nestinþ cells or CM protected mice from renal function impairment and against tubular cell damage after acute injury. MSCs were originally isolated from bone marrow by Friedenstein and colleagues in the 1960s [32]. Over the following decades, MSCs were also found in almost all postnatal organs and tissues, such as adipose tissue [33], heart [34], teeth [35], liver [36], umbilical cord [37] and so on. Importantly, several evidences demonstrated that there were MSCs also existing in kidney [9,38]. However, there were limited specific markers to identify and isolate the renal MSCs. Here, we prospectively isolated renal Nestinþ cells from kidney basing the GFP fluorescence intensity of Nestin-GFP mice. Like as other MSCs populations, renal Nestinþ cells were capable of long-term growth in vitro, and could form clone by single cell and kept proliferation capacity even growing to passage 25. Both the cloned and noncloned cells owned multipotency, and could differentiate into

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further verify the potential renal-protective role of VEGF from Nestinþ cells, VEGF expression in Nestinþ cells was disrupted by small RNA interference. VEGF siRNA highly effectively knocked down VEGF expression (90%) compared with the NTC through ELISA analysis (P < 0.05) (Fig. 7B). When VEGF expression was disrupted, CM could not significantly decreased serum creatinine and BUN levels at day 4 compared with NTC group (Fig. 7C, D). Meanwhile, the protective effect of CM from Nestinþ cells on tissue damage, including necrosis, fibrosis and apoptotic cell death was also abrogated when disrupting VEGF expression (Fig. 7EeG). These data indicate that VEGF secretion may be essential for Nestinþ cells protecting mice from renal function impairment. Thus, these results suggested that Nestinþ cells likely mediated their therapeutic effect partially through secreting VEGF.

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Fig. 6. Nestinþ cells-CM promotes functional recovery in acute ischemic renal injury. (A, B) The treatment with the CM of Nestinþ cells significantly reduced the serum creatinine and BUN levels at 4 days after injury. (Data are expressed as the mean ± SEM; (*P < 0.05). (C, D) Representative photomicrograph showed H&E and Masson stained renal sections in each experimental group at 4 days after injury. Scale bars: 50 mm.

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Fig. 7. Nestinþ cells exert their protective effects on mice with acute ischemic renal injury via VEGF. (A) The mRNA expression of cytokine factors in Nestinþ cells. (B) Transfection of cells with VEGF siRNA significantly decreased VEGF production as seen by ELISA (*P < 0.05). (C, D) Comparison of serum creatinine and BUN levels in DMEM/F12, NTC-CM and siVEGF-CM treated mice at 4 days after renal ischemia. Data are expressed as the mean ± SEM; *P < 0.05. (E, F) Representative images showed H&E and Masson staining at 4 days after injury. Lesions in kidney of si-VEGF-CM-injected mice were more pronounced comparable to those found in mice treated with NTC-CM. Scale bars: 50 mm. (G) Histological assessment of apoptotic cell death was tested by TUNEL staining in each experimental group. Scale bars: 50 mm.

classical mesenchymal cells, including osteocytes, adipocytes and cartilage cells [39]. The results implied that renal Nestinþ cells might represent a potential MSCs population. Until now, there have been several accepted bio-markers to identify and define MSCs among different tissues or cells [40,41]. In this study, renal Nestinþ cells expressed some mesenchymal markers, such as Sca-1, CD44, CD106, but lack of expression CD45 and CD11b by FACS analysis. By immunofluorescent staining, these cells co-expressed Nestin with vimentin [42], NG2 [43] and PDGFRa [27], which were all reported as potential markers of MSCs. Except these markers, renal Nestinþ cell also expressed the embryonic organ-specific marker Pax2. In the developing kidney, Pax2 proteins are among the earliest markers for the renal epithelial cell

lineage, with expression in the mesenchyme and in proliferating epithelia [44]. In addition, these cells also could differentiate into podocytes in the vitro and vivo. Taken together, our results suggested that Nestinþ cells were renal resident MSC-like population. Nestin is highly expressed in renal interstitial cells [45,46] or podocytes [47,48] after suffering from tubulointerstitial injury or Glomerular Disease. These evidences suggested that renal Nestin expressing cells may have important roles in kidney homeostasis and tissue repair. In our study, renal Nestinþ cells could ameliorate the damaged kidney and promote functional recovery after acute ischemic kidney injury as decreasing the peak level of serum creatinine and BUN, and reducing the damaged cell apoptosis. Interestingly, along with the improvement of renal function after

M.H. Jiang et al. / Biomaterials 50 (2015) 56e66

Nestinþ cells infusion, we did not observe obvious integration of these cells into kidney tubules, interstitial and glomeruli, suggesting Nestinþ cells might participate to renal tissue repair through paracrine action. Previous studies have demonstrated that MSC CM exerted a beneficial effect on renal function and tubular damage [49,50]. Also, the secretion of bioactive factors is thought to play a critical role in MSC-mediated immune/inflammatory suppression and paracrine activity, such as HGF [51], VEGF [52], IGF-1 [5], FGF2 [53] and G-CSF. Among these factors, VEGF could mediate endothelial and epithelial cell proliferation and survival after renal injury [54]. Disrupting VEGF expression reduced the effectiveness of rat bone marrow MSC on renal functional recovery in ischemic renal injury model [55]. In our study, CM from Nestinþ cells could markedly ameliorate tissue damage, reduced apoptosis and fibrosis. Furthermore, knocking down VEGF expression in Nestinþ cells abrogated their renal protective effects. These results were consistent with previous reports, further support the renal reparative function of Nestinþ kidney resident MSCs through VEGF, although additional studies will be required to elucidate the underlying mechanism. 5. Conclusion The prospectively isolated renal Nestinþ cells exhibited selfrenewal and differentiation properties. These cells may be a population of MSCs resident in the kidney and protect renal cells from death and aid functional recovery after ischemic injury by secreting VEGF. All these results suggest that renal Nestinþ MSCs may be ideal choice for stem cell-based kidney replacement therapy. Acknowledgments This research was supported by the National Basic Research Program of China (2012CBA01302), the National Natural Science Foundation of China (81425016, 81270646, 81200945, 81370214, 31171398, U1134007), the Key Scientific and Technological Projects of Guangdong Province (2007A032100003), the Guangdong Natural Science Foundation (S2013030013305), the Guangzhou Science and Technology Funds (201400000003-3, 201300000089, 2010U1E00551), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (GDUPS, 2013); Guangdong Department of Science & Technology Translational Medicine Center grant (2011A080300002). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.01.029. References [1] Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2008;2:313e9. [2] Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood 2007;110:3499e506. [3] Baer PC, Geiger H. Mesenchymal stem cell interactions with growth factors on kidney repair. Curr Opin Nephrol Hypertens 2010;19:1e6. [4] Tan J, Wu W, Xu X, Liao L, Zheng F, Messinger S, et al. Induction therapy with autologous mesenchymalstem cells in living-related kidney transplants: a randomizedcontrolled trial. JAMA 2012;307:1169e77. [5] Imberti B, Morigi M, Tomasoni S, Rota C, Corna D, Longaretti L, et al. Insulinlike growth factor-1 sustains stem cell mediated renal repair. J Am Soc Nephrol 2007;18:2921e8. [6] Westenfelder C, Togel FE. Protective actions of administrated mesenchymal stem cells in acute kidney injury: relevance to clinical trials. Kidney Int Suppl 2011;1:103e6. [7] da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 2006;119:2204e13.

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