Kidney Tissue Reconstruction by Fetal Kidney ... - Wiley Online Library

TISSUE-SPECIFIC STEM CELLS Kidney Tissue Reconstruction by Fetal Kidney Cell Transplantation: Effect of Gestation Stage of Fetal Kidney Cells SANG-SOO KIM,a SO-JUNG GWAK,b JOUNGHO HAN,c HEUNG JAE PARK,d MOON HYANG PARK,e KANG WON SONG,e SEUNG WOO CHO,a YUN HEE RHEE,f HYUNG MIN CHUNG,g BYUNG-SOO KIMa a

Department of Bioengineering and bDepartment of Chemical Engineering, Hanyang University, Seoul, Korea; Department of Pathology and dDepartment of Urology, School of Medicine, Sungkyunkwan University, Seoul, Korea; eDepartment of Pathology, College of Medicine, Hanyang University, Seoul, Korea; fChabiotech Co., Ltd., Seoul, Korea; gCHA Stem Cell Institute, Pochon CHA University, Seoul, Korea c

Key Words. Fetal kidney cells • Gestation stage • Kidney failure • Kidney tissue reconstruction

ABSTRACT Dialysis and kidney transplantation, current therapies for kidney failure, have limitations such as severe complications, donor shortage, and immune-related problems. The development of an alternative treatment for kidney failure is demanded. The present study shows that the transplantation of fetal kidney cells reconstitutes functional kidney tissue, and that the gestation stage of kidney cells influences the kidney reconstitution. Fetal kidney cells were isolated from metanephroi of rat fetuses at various gestation stages and transplanted into the omentum or kidney of immunodeficient mice. Immunophenotype analysis of fetal kidney cells showed apparent expression of stem cell markers. Three weeks after transplantation, histological analyses of retrieved grafts revealed the formation of kidney structures, including fluorescently labeled transplanted cells, suggesting

the potential of fetal kidney cells to reconstitute kidney tissues. The grafts retrieved from omentum contained cystic fluids with concentrated solutes. However, transplanted early fetal kidney cells had also differentiated into nonrenal tissues such as bone and cartilage. In addition, transplantation of fetal kidney cells from a later gestation stage resulted in poor kidney structure formation. Kidney-specific genes were strongly expressed in the earlier cell transplants. The cells at an earlier gestation stage had higher colony forming ability than the cells at a later stage. This study demonstrates the reconstitution of kidney tissue by transplanting fetal kidney cells and the presence of an optimal time window in which fetal kidney cells regenerate kidney tissues. STEM CELLS 2007;25: 1393–1401

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION End-stage renal disease (ESRD) is a destructive and devastating disease caused by a progressive and irreversible loss of functioning nephrons in the kidney. Although dialysis is life-saving and prolongs survival for many patients with ESRD, it is only temporary and does not replace all of the kidney’s functions [1]. Allogeneic kidney transplantation is the only current means to restore the whole kidney function, but its application is severely limited by donor shortage and immune-related problems [2– 4]. Developing kidney precursors (metanephroi) isolated from early embryos and fetuses may be a potential source for regenerating kidney tissue [5, 6]. Previously, growing new kidneys in situ via the transplantation of developing kidney precursor tissues isolated from embryos has been studied [7–10]. In one study, embryonic kidney tissues obtained from early human and porcine embryos and transplanted into immunodeficient mice underwent nephrogenesis and exhibited partial excretory function [8]. However, cell transplantation may be advantageous over transplantation of a whole vascularized organ, since the former is less susceptible to humoral rejection [11]. Kidney cells isolated from cloned bovine fetuses, seeded on polymer matrices,

and transplanted into the nuclear donor animal partially regenerated kidney structures that excreted urine-like fluid in vivo [12]. However, the utilized fetal kidney tissues were different in species and gestation stages among previous studies and the influences of gestation stages on regeneration have not yet been reported. In the present study, we investigated whether the transplantation of fetal kidney cells is capable of regenerating functional kidney tissues in vivo and whether the gestation stage of the transplanted fetal kidney cells influences kidney tissue reconstitution. The fetal kidney cells were isolated from rat metanephroi, seeded onto three-dimensional matrices, and implanted into the omentum or kidney of immunodeficient mice. The transplanted cells organized into glomerular and tubular structures 3 weeks after transplantation, which was strongly dependent on the gestation stage of the fetal kidney cells.

MATERIALS

AND

METHODS

Isolation of Fetal Kidney Cells Whole types of fetal kidney cells were isolated from the metanephroi of Sprague-Dawley (SD) rat (SLC, Tokyo, http://www.jslc.

Correspondence: Byung-Soo Kim, Ph.D., Department of Bioengineering, Hanyang University, 17 Haengdang-dong, Seongdong-ku, Seoul 133-791, Korea. Telephone: ⫹82-2-2220-0491; Fax: ⫹82-2-2291-0838; e-mail: [email protected] Received March 28, 2006; accepted for publication February 15, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2006-0183

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co.jp) fetuses at embryonic days 14.5 (E14.5; plug day ⫽ 0), 17.5 (E17.5), and 20.5 (E20.5) as described previously [13]. In brief, fetal kidneys were surgically dissected from fetuses of SD rats under a microscope. Isolated metanephroi were washed with cold Hanks’ balanced saline solution (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) containing 10 mM N-2-hydroxyethylpiperazine-N⬘-2-ethane sulfonic acid (Gibco-BRL) and were transferred to cold Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL) containing 10% (vol/vol) fetal bovine serum (FBS) (Gibco-BRL) and antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin; Gibco-BRL). Transferred metanephroi were minced into small pieces and digested in a 1-mg/ml collagenase/dispase (Roche Diagnostics, Basel, Switzerland, http://www.roche-appliedscience.com) solution at 37°C for 60 minutes with continuous shaking using an orbital shaker. After 60 minutes of digestion, single cells were obtained by filtering the digested tissue through 40-␮m nylon mesh (Cell Strainer; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) to remove undigested glomerular and tubular structures. After this step, we confirmed that only single cells were obtained by microscopic examination. The strained cells were collected by centrifugation. The number of total cells isolated from kidney tissue varies depending on the gestation time of the kidney. An equal number of cells for all gestation stages was transplanted for the kidney tissue regeneration study. Adult kidney cells were cultured from SD rats (8-week-old, male) with a similar procedure.

Fluorescence-Activated Cell Sorter Analyses For surface antigen phenotyping, singly isolated fetal kidney cells and adult kidney cells were rinsed and incubated in 2% (vol/vol) FBS/phosphate-buffered saline buffer containing either mouse IgG and IgM for reference or predetermined concentrations of fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated antibodies at 4°C for 30 minutes. Washed samples were analyzed with FACSVantage SE (Becton Dickinson) using the CellQuest software (Becton Dickinson). The antibodies used were c-kit-APC, Oct-4-PE, SSEA-1-PE (R&D Systems Inc., Minneapolis, http://www.rndsystems.com); CD133-APC (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec. com); CD34-PE, CD73-PE, CD90-PE (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml); and CD105-FITC (Chemicon, Temecula, CA, http://www.chemicon.com). As isotypic control, mouse IgM-FITC (Chemicon), mouse IgG1-PE, IgM-PE, rat IgG2B-PE, and mouse IgG-APC (BD Pharmingen) were used.

Cell Labeling Fetal kidney cells were fluorescently labeled with enhanced green fluorescence protein (eGFP) prior to transplantation. The MMP-f2IRES-eGFP retroviral vector was introduced into 293gpg retrovirus packaging cell line by transient transfection with Lipofectamine (Gibco-BRL). After 72 hours, the supernatants were harvested and infected with polybrene (1 ␮g/ml) for 2 hours [14]. Isolated fetal kidney cells were incubated in DMEM containing 10% (vol/vol) fetal bovine serum for 1 day. The incubated cells (unpassaged) were further incubated with the viral supernatant for 1 day prior to transplantation [15].

Scaffolds for Cell Transplantation Porous nonwoven mesh fabricated from polyglycolic acid (PGA), a biodegradable synthetic polymer, fibers (12 ␮m in diameter; Albany International Inc., Mansfield, MA, http://www.albint.com) were utilized as three-dimensional scaffolds for cell transplantation to omentum. One day prior to use, the scaffolds were cut into squares (5 ⫻ 5 mm2, 2 mm in thickness), sterilized with ethanol, and washed with sterile distilled water. The sterilized scaffolds were then soaked in DMEM containing 10% (vol/vol) FBS until use. Fibrin gel matrix, a vehicle for cell transplantation to the kidney, was prepared from a commercially available fibrin gel kit (Greenplast; GreencrossPD Co., Yongin, Korea, http://www. greencross.com). Plasminogen-free fibrinogen (100 mg) and fibrinstabilizing factor XIII (66 units) were dissolved in 1 ml of plasmin inhibitor aprotinin solution (1,100 kIU/ml) for fibrinogen solution.

Fetal Kidney Cell Transplantation Thrombin (500 IU) was dissolved in 1 ml of calcium chloride solution (5.9 mg/ml) for thrombin solution. For gelation, the thrombin solution containing cells and fibrinogen solution was mixed in situ at a 1:1 volume ratio. Fibrin gel is formed when fibrinogen is activated by thrombin in the presence of Ca2⫹ ion and factor XIII.

Transplantation of Fetal Kidney Cells into the Omentum of Immunodeficient Mice PGA scaffolds were used as three-dimensional matrices to transplant fetal kidney cells into the omentum of immunodeficient mice (BALB/c-nu, 7-week-old female; SLC). Isolated fetal kidney cells were suspended in DMEM at a concentration of 2.07 ⫻ 107 cells per milliliter. A cell suspension of 0.05 ml was seeded onto each PGA scaffold [16]. To ensure cell attachment to the PGA scaffolds, the cell-seeded scaffolds were incubated in a humidified 5% CO2 atmosphere for 30 minutes prior to transplantation. The cell-seeded scaffolds were then transplanted into the omentum of immunodeficient mice (n ⫽ 8 for each group). After the mice were anesthetized with an intramuscular administration of ketamine hydrochloride (50 mg/kg; Yuhan Co., Seoul, Korea, http://www.yuhan.co.kr/Eng Main.asp) and xylazine hydrochloride (5 mg/kg; Bayer Korea Ltd., Seoul, Korea, http://www.bayer.co.kr/eng/index.asp), a midline laparotomy was performed. The cell-seeded matrices were transplanted into the omentum and the wounds were closed with a 5-0 suture (VICRYL; Ethicon, Somerville, NJ, http://www.ethicon.com). The mice were singly housed in microisolator cages in a pathogen-free barrier facility under a 12-hour light/dark cycle after surgery. Food and water were supplied ad libitum. The animals received humane care and all animal experiments were carried out according to the Hanyang University Guide for the Care and Use of Laboratory Animals. Three weeks after transplantation, the grafts were retrieved for analyses.

Transplantation of Fetal Kidney Cells into the Kidney of Immunodeficient Mice Fetal kidney cells were transplanted into the subcapsular and cortex region of the kidneys of immunodeficient mice using fibrin gel matrix as a three-dimensional matrix. Solutions of thrombin (0.5 ml) and fibrinogen (0.5 ml) containing fetal kidney cells (6.0 ⫻ 107 cells) were prepared [17]. The mice were anesthetized, and the kidney was exposed by a midline laparotomy. Through a device designed for simultaneous injection of the fibrinogen and thrombin solutions, 0.05 ml of the fibrin mixture containing fetal kidney cells was injected into the subcapsular and cortex region of the left kidneys of immunodeficient mice (n ⫽ 8 for each group). The grafts were retrieved for analyses 3 weeks after transplantation.

Analyses of Cystic Fluid Cystic fluids were collected into small plastic tubes from the cysts of the explanted E14.5 (n ⫽ 8) and E17.5 (n ⫽ 5) transplants to the omentum of immunodeficient mice 3 weeks after transplantation. The concentrations of urea nitrogen (UN) and creatinine were measured using standard techniques [18]. The levels of UN and creatinine of the serum, peritoneal fluid, and urine samples were measured as controls (n ⫽ 8 for each control group).

Semiquantitative Reverse Transcription-Polymerase Chain Reaction The grafts were harvested three weeks after transplantation to omentum, pulverized in liquid nitrogen, and homogenized in 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen. com). Total RNA was extracted with 0.2 ml of chloroform and precipitated with 0.5 ml 80% (vol/vol) isopropanol. After the removal of the supernatant, the RNA pellet was washed with 75% (vol/vol) ethanol, air-dried, and dissolved in RNase-free water. Complementary DNA was synthesized from 2 ␮g RNA using SuperScript II reverse transcriptase (Invitrogen) and random hexamer primers at 42°C for 60 minutes. Synthesized cDNA was amplified with a thermal cycler (GeneAmp PCR System 2700; Applied BioSystems, Foster City, CA, http://www.appliedbiosystems. com) using the primers in Table 1. Polymerase chain reaction (PCR)

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Table 1. Oligonucleotide primers used for reverse transcription-polymerase chain reaction and predicted product sizes Targets

AQP1 AQP2 THP SYN NEP OC GAPDH

Sequences

F: 5⬘-GTC CCA CAT GGT CTA GCC TTG TCT G-3⬘ R: 5⬘-GGG AAG GGT CCT GGA GGT AAG TCA-3⬘ F: 5⬘-GCC CCT TGC AGG AAC CAG ACA-3⬘ R: 5⬘-GCC AAA GCG GGA ATG ACA GTC-3⬘ F: 5⬘-CTG GAT GTC CAT AGT GAC TC-3⬘ R: 5⬘-TGT GGC ATA GCA GTT GGT CA-3⬘ F: 5⬘-GCA GAG GAA GTG AGG TCC AG-3⬘ R: 5⬘-GAT GCC ACT AGG GTG CTA GG-3⬘ F: 5⬘-GTT CAG CTG GGA GAG ACT GG-3⬘ R: 5⬘-AAT CGG ACG ACA AGA CGA AC-3⬘ F: 5⬘-ATG TGC CCT CCT GGT TCA TTT CTT-3⬘ R: 5⬘- GTG GTC CGC TAG CTC GTC ACA ATT-3⬘ F: 5⬘-CCA GCA GAA ATA TGA CAA CTC CCT C-3⬘ R: 5⬘-GGT GGT GAA GCA GGC GGC CGA GGG-3⬘

Product sizes (bp)

References

362

关25兴

277

关25兴

399

关26兴

173

关27兴

192

关27兴

672

关28兴

348

关29兴

Abbreviations: AQP, aquaporin; bp, base pairs; F, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NEP, nephrin; OC, osteocalcin; R, reverse; SYN, synaptopodin; THP, Tamm-Horsfall protein.

was performed under the following conditions: up to 35 cycles of denaturing at 95°C for 30 seconds, annealing at 56°C for 30 seconds (aquaporin [AQP]1, AQP2, synaptopodin [SYN]), 55°C for 60 seconds (Tamm-Horsfall protein [THP], nephrin [NEP], glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), 60°C for 30 seconds (osteocalcin [OC]), and extension at 72°C for 60 seconds; this was followed by a final extension at 72°C for 7 minutes. The PCR products were visualized with 2.0% (wt/vol) agarose gel electrophoresis and ethidium bromide staining. Gel readings and densitometric analyses were carried out using a gel documentation system (Gel Doc 1000; Bio-Rad, Hercules, CA, http://www.bio-rad.com). The expression level of each mRNA was normalized using the GAPDH housekeeping gene product as an endogenous reference.

Histological and Immunohistochemical Analyses For histological analyses, the partial tissue samples of the retrieved specimens were fixed in 10% (vol/vol) buffered formalin, dehydrated in ascending grades of ethanol, and embedded in paraffin. We processed 5-␮m-thick tissue sections for H&E staining and Masson’s trichrome staining. Glomeruli were counted under a microscope. For immunohistochemical analyses, 5-␮m-thick sections were stained using antibodies against proliferating cell nuclear antigen (PCNA) (DAKO, Glostrup, Denmark, http://www.dako. com). The staining signals were developed using a streptavidin biotin universal detection system (UltraTech HRP; Immunotech, Luminy, France, http://www.beckmancoulter.com/products/pr_ immunology.asp) and 3,3⬘-diaminobenzidine tetrahydrochloride substrate solution (Vector Laboratories, Burlingame, CA, http:// www.vectorlabs.com). Mayer’s hematoxylin (ScyTek, Logan, UT, http://www.scytek.com) was used for counterstaining.

Fluorescence Microscopy Frozen tissue sections were used to observe the fluorescence in the retrieved transplants. Partial tissue samples were fixed in 10% (vol/vol) buffered formalin and processed for routine pathology. The remaining tissues were embedded in Tissue-Tek OCT compound (Sakura, Torrance, CA, http://www.sakuraus.com), snap frozen in liquid nitrogen, and stored at ⫺70°C until use. We stained 5-␮m-thick sections using antibodies against anti-cytokeratin (DAKO), then stained with Texas Red conjugated goat anti-rabbit IgG (red fluorescence; Molecular Probes, Carlsbad, CA, http:// probes.invitrogen.com). Stained tissue sections were analyzed for eGFP labeling (green fluorescence) under a fluorescent microscope (Olympus BX51; Olympus America Inc., Melville, NY, http:// www.olympusamerica.com) 3 weeks after transplantation into the kidneys of immunodeficient mice. Tissue sections were also counterstained and mounted in VECTASHIELD Mounting Medium with 4 – 6-diamidino-2-phenylindole (Vector Laboratories).

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Colony-Forming Unit Assay For the measurement of colony-forming units, cells were suspended at a density of 2.3 ⫻ 104 cells per milliliter in methylcellulose (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich. com). We plated 1-ml aliquots of cell suspension in triplicate in gridded 35-mm dishes (Nunc, Rochester, NY, http://www. nuncbrand.com) and incubated for 2 weeks under fully humidified conditions in an atmosphere of 5% O2 and 5% CO2 at 37°C [19]. Colonies with more than 50 cells were scored with a dark-field stereomicroscope.

Statistical Analyses Quantitative data were expressed as the mean ⫾ SD. Statistical comparisons were carried out using analysis of variance (SAS Institute Inc., Cary, NC, http://www.sas.com). The data were considered statistically significant when p ⬍ .05.

RESULTS Characterization of Fetal Kidney Cell Surface Phenotype Examination of the immunophenotype of fetal kidney cells revealed the apparent populations of stem cells in the fetal kidneys. The fetal kidney cells have cell fractions expressing CD73 (5⬘ terminal nucleotidase/SH-3), CD90 (thy-1), and CD105 (endoglin/SH-2) (mesenchymal surface markers); CD34, CD133, and c-kit (hematopoietic or hemangioblastic surface markers); and Oct-4 and SSEA-1 (embryonic surface markers) on their surfaces (Fig. 1A–1C). In contrast to fetal kidney cells, adult kidney cells had negligible expression of these surface markers, with the exception of CD90, which was detected at a much lower level than that of fetal kidney cells (Fig. 1D). The expression patterns of stem cell surface markers varied depending on the gestation stages. As the gestation stage proceeds, the expressions of mesenchymal and embryonic surface markers decreases, as indicated by the fact that whereas more than 60% of the E14.5 fetal kidney cells were SSEA-1⫹ and Oct-4⫹, 30%– 45% of the E20.5 fetal kidney cells were SSEA-1⫹ and Oct-4⫹ (Fig. 1E). However, the expressions of hematopoietic surface markers increased in E17.5 fetal kidney cells and then decreased after that stage (Fig. 1E).

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Fetal Kidney Cell Transplantation

Figure 1. Immunophenotype characterization of kidney cells. Kidney cells were isolated from (A) E14.5, (B) E17.5, (C) E20.5, and (D) adult kidney. Shown is the expression of respective antigens (green, red, or pink lines, detected with FITC-, PE-, or APC-conjugated antibodies, respectively) on kidney cells together with isotype controls (black line). (E): Ratio of kidney cell populations positive for the surface markers. Abbreviations: APC, allophycocyanin; E, embryonic day; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Transplantation of Fetal Kidney Cells into the Omentum of Immunodeficient Mice Three weeks after transplantation, the transplanted E14.5 and E17.5 fetal kidney cells formed three-dimensional kidney tissues with extensive vascularization (Fig. 2A, 2D). All E14.5 transplants and five out of eight E17.5 transplants contained cystic fluids. The volumes of the cystic fluids were approximately 50 –200 ␮l. Histological analysis of the E14.5 transplants showed early kidney structures and maturing nephrons, including developing kidney tubules and glomeruli with well formed vascular tufts (Fig. 2B). However, the transplanted early fetal (E14.5) kidney cells had also differentiated into nonrenal tissues such as bone and cartilage (Fig. 2C). The transplantation of fetal kidney cells at later stages of gestation (E17.5 and E20.5) also resulted in three-dimensional tissue formation. However, histo-

logical analysis of the E20.5 transplants revealed fewer tubular and glomerular structures (Fig. 2F) than in the E14.5 and E17.5 transplants (Fig. 2B, 2E). Nonrenal tissue formation was not observed in the E17.5 and E20.5 transplants. To determine the functionality of the reconstituted kidney tissues, the cystic fluids from the E14.5 and E17.5 transplants were chemically analyzed. The UN levels in the cystic fluids were 73.63 ⫾ 20.20 and 54.67 ⫾ 7.51 mg/dl in the E14.5 and E17.5 transplants. The UN levels were 30.18 ⫾ 1.90, 38.04 ⫾ 4.58, and 3,058.33 ⫾ 283.51 mg/dl in the serum, peritoneal fluid, and urine, respectively. The creatinine levels in the cystic fluids were 2.47 ⫾ 0.64 and 1.63 ⫾ 0.51 mg/dl in the E14.5 and E17.5 transplants. The creatinine levels were 0.30 ⫾ 0.03, 0.47 ⫾ 0.11, and 37.13 ⫾ 2.90 mg/dl in the serum, peritoneal fluid, and urine, respectively. Although the levels of UN and creatinine in the cystic fluids were much lower than those in the


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Figure 2. Transplants retrieved from the omentum of immunodeficient mice 3 weeks after transplantation. (A, D): Macroscopic views of (A) E14.5 and (D) E17.5 transplants. Scales are in centimeters. The transplants contained large cysts (ⴱ). (B): Histological analysis (H&E staining) identified primitive tubules (white arrow), glomeruli (black arrow), newly formed blood vessels (white arrowhead), and polyglycolic acid fibers (black arrowhead) in the E14.5 transplants. (C): E14.5 cells partially differentiated into bone (†) and cartilage (‡). (E): The retrieved E17.5 transplants showed the formation of tubules and glomeruli, (F) but the E20.5 transplants showed very little kidney tissue formation (scale bars ⫽ 20 ␮m). Analyses of the cystic fluids from the explanted tissue specimens 3 weeks after transplantation into the omentum of immunodeficient mice for (G) urea nitrogen and (H) creatinine. E14.5 (n ⫽ 8) and E17.5 (n ⫽ 5). #, p ⬍ .05 compared with serum, peritoneal fluid, and urine. Abbreviation: E, embryonic day.

urine samples of the recipient mice, they were significantly higher than those in the serum and peritoneal fluid (Fig. 2G, 2H). The mRNA of kidney-specific genes AQP1 (specific marker of proximal tubule and descending loop of Henle), AQP2 (specific marker of collecting duct), THP (specific marker of distal tubule), NEP (specific marker of glomerular podocytes), and SYN (specific marker of glomerular visceral epithelial cell) was strongly expressed in the E14.5 and E17.5 transplants, but mRNA of these kidney-specific genes was weakly expressed in the E20.5 transplants (Fig. 3). The expression of OC, a specific marker of bone, was observed in the E14.5 transplant but not in the E17.5 or E20.5 transplants.

Transplantation of Fetal Kidney Cells into the Kidney of Immunodeficient Mice When E14.5 fetal kidney cells were transplanted into the kidney using a fibrin gel matrix, the cells survived and reconstituted primitive kidney structures, including glomeruli and tubules, 3 weeks after transplantation. More glomeruli and tubules with more mature structures were observed in the reconstituted regions compared with those resulting from cell transplantation into the omentum (Fig. 4B– 4D). Histological examination of the E14.5 transplants showed early kidney structures with comma- or S-shaped bodies and maturing nephrons, including primitive tubules and primitive glomeruli with vascular tufts (Fig. 4A, 4B). A prominent visceral epithelial layer characteristic of fetal glomeruli was noted (Fig. 4D). The formation of nonrenal www.StemCells.com

tissue such as bone and cartilage was also observed (Fig. 4A– 4C). The transplantation of E17.5 fetal kidney cells into the kidneys of immunodeficient mice resulted in the reconstitution of more mature kidney structures. These were more similar to those of normal nephrons (Fig. 4E– 4G) than those from the transplantation of E14.5 cells. Retrieved transplants showed distinguishable reconstituted regions between the normal regions of the kidney (Fig. 4E, 4F). In the reconstituted region, newly formed tubular and glomerular structures were observed, and the density of glomeruli was greater than in a normal kidney (Fig. 4F). The newly formed glomeruli (Fig. 4H) were structurally similar to normal glomeruli. Nonrenal tissue formation was not observed in the E17.5 transplants. In contrast, the E20.5 transplants showed poor kidney development (Fig. 4I). The E20.5 fetal kidney cells reconstituted few glomerular and tubular structures. To investigate the fate of the transplanted fetal kidney cells, the cells were labeled with eGFP prior to transplantation. The fetal kidney cells expressing eGFP (green fluorescence) were detected in the reconstituted kidney tubules, which were stained positively for anticytokeratin (red fluorescence) in the E17.5 transplants at 3 weeks (Fig. 5A). Immunohistochemical analyses using antibodies against PCNA, a marker of proliferating cells, showed that PCNApositive cells were more abundantly observed in the transplants of earlier fetal kidney cells (Fig. 6A, 6B) than in the transplants of fetal kidney cells from later gestation stages (Fig. 6C). PCNA-positive cells were very rarely seen in the normal kidney tissues (Fig. 6D). The intensive proliferative activity may be


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Figure 3. Semiquantitative reverse transcription-polymerase chain reaction analysis for AQP1, AQP2, NEP, SYN, THP, and OC expression in E14.5 (●), E17.5 (E), and E20.5 () transplants retrieved from the omentum of immunodeficient mice 3 weeks after transplantation (n ⫽ 3 for each group). GAPDH was used as a housekeeping mRNA control. §, p ⬍ .05 between the E14.5 and E17.5 transplants. ¶, p ⬍ .05 between the E17.5 and E20.5 transplants. Abbreviations: AQP, aquaporin; E, embryonic day; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NEP, nephrin; OC, osteocalcin; SYN, synaptopodin; THP, Tamm-Horsfall protein.

related to the stem cell characteristics of the kidney precursor cells present in the mesenchyme of the metanephros [20]. Similarly, in vitro colony formation ability was higher in the E14.5 and E17.5 cells than in the E20.5 fetal kidney cells (Fig. 6E). The cells in the colony assay exhibited fibroblast-like and stromal morphology.

DISCUSSION Our results show that transplanted fetal rat kidney cells can survive and undergo nephrogenesis, forming kidney structures capable of producing fluid with increased concentrations of UN and creatinine. The kidney regeneration capability was dependent on the gestation stage of the fetal kidney cells. In the present study, fetal kidney cells were utilized as a cell source for kidney tissue reconstitution instead of differentiated adult kidney cells; that is because fetal kidney cells may have a higher ability to differentiate into all types of kidney cells than adult kidney cells and may be more effective for kidney tissue reconstitution. As the kidney has extremely complex structures and functions [21], the cellular components are very complicated, and more than 28 types of terminally differentiated cells are present in the adult kidney. Therefore, it may not be feasible

to regenerate kidney tissues in vivo by transplanting various types of terminally differentiated adult kidney cells. When we transplanted adult (8-week-old) rat kidney cells, we did not observe kidney tissue formation (data not shown). In contrast, the fetal kidney cells, which can differentiate into all types of cells present in the kidney [20], may regenerate the kidney efficiently when transplanted. The surface phenotype analyses of fetal kidney cells in this study showed the apparent populations of stem cells in the fetal kidneys that are positive for mesenchymal, hematopoietic, and embryonic surface markers. In addition, the formation of nonrenal tissues, such as bone and cartilage, in the early fetal kidney cell transplants indicates that fetal kidneys in the early gestation stages contain several types of committed progenitor or stem cells, and these cells have the ability to generate nonrenal cell types [10, 20]. Therefore, the transplantation of developing fetal kidney cells might be advantageous over the transplantation of mature adult kidney cells in regenerating kidney tissue. Although nephrons have previously been grown in vivo from fetal kidney tissue [22, 23], no study has shown in vivo structural reconstitution of kidney tissue by fetal kidney cell transplantation. Fetal kidney cells isolated from rat fetuses were used to generate kidney tissue in the present study. The recon-


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Figure 4. Retrieved transplants from the kidneys of immunodeficient mice 3 weeks after transplantation. (A–C): In the E14.5 transplants, the formation of nonrenal tissue, such as cartilage (‡) and bone (†), was observed. (D): A prominent visceral epithelial layer (dotted line) was noted in the developing glomeruli (black arrow) in E14.5 transplants. (E–G): In the E17.5 transplants, kidney tissue formation was observed. (H): The newly formed glomeruli were structurally similar to normal glomeruli. (I): E20.5 transplants showed poor kidney tissue formation. Scale bars ⫽ 5 ␮m (D, H), 20 ␮m (B, C, G, I), 50 ␮m (A, F), and 200 ␮m (E).

Figure 5. Using fluorescence (eGFP) labeling, the transplanted fetal kidney cells were detected in the reconstituted kidney tubules (arrows) of the E17.5 transplant 3 weeks after transplantation. (A): A merged image of cytokeratin (red), eGFP (green), and DAPI (blue). (B): Matching H&E staining of (A). Scale bars ⫽ 25 ␮m. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein.

stituted kidney structures contained developing glomeruli and tubules, which are similar to those of developing kidneys undergoing nephrogenesis. The regenerated kidney tissues also had a fluid with concentrated UN and creatinine. Although the concentrations of UN and creatinine were much lower than that of urine, the produced fluid showed concentrated UN and creatinine levels compared with those of serum and peritoneal fluid. In this study, we have used biocompatible and biodegradable polymer (PGA) meshes or polymer gel (fibrin gel) as three-dimensional scaffolds for kidney tissue reconstitution. These scaffolding materials provide cells with surfaces to attach to and spaces to regenerate new tissues in. In addition, degradation of these biodegradable scaffolds would allow for the removal of synthetic parts from the implants and the subsequent formation of natural tissues. We have used two different transplantation models, ectopic (omentum) and orthotopic (kidney), to investigate the nephrogenic potential of fetal kidney cells. Cell transplantation into the ectopic www.StemCells.com

site clearly shows that fetal kidney cells can reconstitute kidney tissues by themselves, as evidenced by the formation of kidney structures including glomerular and tubular structures, and that the gestation stage of the transplanted fetal kidney cells affects nephrogenic development. The study on fetal kidney cell transplantation to the orthotopic site shows the possibility that cells transplanted to impaired kidney can augment kidney tissue. Cells transplanted into the orthotopic site reconstituted more mature kidney structures compared with those transplanted into ectopic site. The gestation stages of transplanted fetal kidney cells affected the regeneration of kidney tissues. Successful organogenesis in the kidney was achieved when the earlier fetal kidney cells were transplanted, but not when kidney cells from a later gestation stage (E20.5) were used. However, fetal kidney cells at earlier stage (E14.5) failed to differentiate exclusively into the desired professional cell fate; instead, they also formed nonrenal differentiated derivatives. Therefore, there exists an optimal time window of gestation stages that can regenerate the kidney tissue efficiently without the formation of nonrenal tissues. These results are consistent with previous reports that stem cells or progenitor cells may have a time window when they exert their beneficial effects on tissue regeneration [24]. It may be due to the fact that the number and stemness of the committed progenitor or stem cells in developing metanephroi of fetuses may vary depending on the gestation stage. Our results of immunophenotype analyses also indicate decreased populations of the committed progenitor or stem cells at later gestation stage. There would also exist the genes that dictate the fetal kidney cells to differentiate into nephrogenic pathway. These genetic cues would be important to understand the mechanism of gestational time-dependent regeneration of the kidney. However, we could not perform the study to understand the mechanism because no genetic markers that induce nephrogenic differentiation have been found yet. This study shows the possibility of transplanting fetal kidney cells as a potential method for partial augmentation of


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Figure 6. The proliferative activity of fetal kidney cells at various gestation stages. Immunohistochemical staining for proliferating cell nuclear antigen in (A) E14.5, (B) E17.5, and (C) E20.5 transplants and (D) normal kidney tissue (scale bars ⫽ 5 ␮m). (E): Colony formation ability of fetal kidney cells at various gestation stages, as examined by the CFU assay (n ⫽ 3). ⴱ, p ⬍ .05. Abbreviations: CFU, colony-forming unit; E, embryonic day.

kidney structures. However, further studies are necessary to assess the therapeutic potential of this method. In order to better understand how fetal kidney cell transplantation exerts kidney tissue regeneration, further studies concerning identification of molecular markers for kidney stem/progenitor cells and the further characterization of the cell population in the fetal kidney cells are required. In addition, cell transplantation studies with selected lineages of fetal kidney cells are needed to understand which types of cells can contribute to and are important for kidney regeneration. Fetal kidney cell transplantation experiments need to be performed in animal models with fully immunocompetent systems, since immunological response may affect the regeneration of kidney structures and functions. It would be critical to investigate whether transplantation of fetal kidney cells to animal models with renal failure can restore kidney functions.

REFERENCES 1

Humes HD, Szczypka MS. Advances in cell therapy for renal failure. Transpl Immunol 2004;12:219 –227. 2 Amiel GE, Atala A. Current and future modalities for functional renal replacement. Urol Clin North Am 1999;26:235–246. 3 Hammerman MR. Xenotransplantation of developing kidneys. Am J Physiol Renal Physiol 2002;283:F601–F606. 4 Kim BS, Mooney DJ, Atala A. Genitourinary system. In: Lanza RP, Langer R, Vacanti J eds. Principles of Tissue Engineering. San Diego: Academic Press, 2000:655– 667. 5 Hammerman MR. Organogenesis of kidneys following transplantation of renal progenitor cells. Transpl Immunol 2004;12:229 –239. 6 Dekel B, Reisner Y. Embryonic committed stem cells as a solution to kidney donor shortage. Expert Opin Biol Ther 2004;4:443– 454. 7 Rogers SA, Hammerman MR. Transplantation of rat metanephroi into mice. Am J Physiol Regul Integr Comp Physiol 2001;280: R1865–R1869. 8 Rogers SA, Lowell JA, Hammerman NA et al. Transplantation of developing metanephroi into adult rats. Kidney Int 1998;54:27–37. 9 Dekel B, Burakova T, Ben-Hur H et al. Engraftment of human kidney tissue in rat radiation chimera: II. Human fetal kidneys display reduced immunogenicity to adoptively transferred human peripheral blood mononuclear cells and exhibit rapid growth and development. Transplantation 1997;64:1550 –1558. 10 Dekel B, Burakova T, Arditti FD et al. Human and porcine early kidney precursors as a new source for transplantation. Nat Med 2003;9:53– 60. 11 Edge AS, Gosse ME, Dinsmore J. Xenogeneic cell therapy: Current progress and future developments in porcine cell transplantation. Cell Transplant 1998;7:525–539. 12 Lanza RP, Chung HY, Yoo JJ et al. Generation of histocompatible tissues using nuclear transplantation. Nat Biotechnol 2002;20:689 – 696.

ACKNOWLEDGMENTS This work was supported by a Grant (number R01-2001-00000491-0) from the Basic Research Program of the Korea Science & Engineering Foundation and a Grant (SC 3220) from the Stem Cell Research Center of the 21st Century Frontier Program funded by the Ministry of Science and Technology, Republic of Korea.

DISCLOSURE

POTENTIAL CONFLICTS OF INTEREST

OF

The authors indicate no potential conflicts of interest.

13 Kim SS, Park HJ, Han J et al. Renal precursor cell transplantation using biodegradable polymer scaffolds. J Microbiol Biotechnol 2005;15: 105–111. 14 Ory DS, Neugeboren BA, Mulligan RC. A stable human-derived packaging cell line for production of high-titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci U S A 1996;93:11400 –11406. 15 Kim JH, Lee JH, Park JY et al. Retrovirally transduced NCAM140 facilitates neuronal fate choice of hippocampal progenitor cells. J Neurochem 2005;94:417– 424. 16 Kim SS, Park HJ, Han J et al. Renal tissue reconstitution by the implantation of renal segments on biodegradable polymer scaffolds. Biotechnol Lett 2003;25:1505–1508. 17 Ryu JH, Kim IK, Cho SW et al. Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium. Biomaterials 2005;26:319 –326. 18 Combet S, Van Landschoot M, Moulin P et al. Regulation of aquaporin-1 and nitric oxide synthase isoforms in a rat model of acute peritonitis. J Am Soc Nephrol 1999;10:2185–2196. 19 Kim BS. Production of human hematopoietic progenitors in a clinicalscale stirred suspension bioreactor. Biotechnol Lett 1998;20:595– 601. 20 Oliver JA, Barasch J, Yang J et al. Metanephric mesenchyme contains embryonic renal stem cells. Am J Physiol Renal Physiol 2002;283: F799 –F809. 21 Al-Awqati Q, Oliver JA. Stem cells in the kidney. Kidney Int 2002;61: 387–395. 22 Woolf AS, Palmer SJ, Snow ML et al. Creation of a functioning mammalian chimeric kidney. Kidney Int 1990;38:991–997. 23 Woolf AS, Hornbruch A, Fine LG. Integration of new embryonic nephrons into the kidney. Am J Kid Dis 1991;17:611– 614. 24 Haller H, de Groot K, Bahlmann F et al. Stem cells and progenitor cells in renal disease. Kidney Int 2005;68:1932–1936. 25 Capurro C, Rivarola V, Kierbel A et al. Vasopressin regulates water flow in a rat cortical collecting duct cell line not containing known aquaporins. J Membr Biol 2001;179:63–70.


26 Arend LJ, Smart A, Briggs JP. Metanephric rat-mouse chimeras to study cell lineage of the nephron. Dev Genet 1999;24:230 –240. 27 Petermann AT, Krofft R, Blonski M et al. Podocytes that detach in experimental membranous nephropathy are viable. Kidney Int 2003;64: 1222–1231. 28 Taira M, Toyosawa S, Ijyuin N et al. Studies on osteogenic differentia-

www.StemCells.com

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tion of rat bone marrow stromal cells cultured in type I collagen gel by RT-PCR analysis. J Oral Rehabil 2003;30:802– 807. 29 Arpornmaeklong P, Kochel M, Depprich R et al. Influence of plateletrich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. Int J Oral Maxillofac Surg 2004;33: 60 –70.