In vitro reconstitution of human kidney structures for renal cell therapy

Article (PDF Available)inNephrology Dialysis Transplantation 27(8):3082-90 · January 2012with15 Reads
DOI: 10.1093/ndt/gfr785 · Source: PubMed
Abstract
Recent advances in cell therapies have provided potential opportunities for the treatment of chronic kidney diseases (CKDs). We investigated whether human kidney structures could be preformed in vitro for subsequent implantation in vivo to maximize tissue-forming efficiency. Human renal cells were isolated from unused donor kidneys. Human renal cells were cultured and expanded. Migration was analyzed using growth factors. To form structures, cells were placed in a three-dimensional culture system. Cells were characterized by immunofluorescence, western blots and fluorescence-activated cell sorting using renal cell-specific markers for podocin, proximal and distal tubules and collecting ducts. An albumin uptake assay was used to analyze function. Three-dimensional cultures were implanted into athymic rat kidneys to evaluate survival. Human renal cells were effectively expanded in culture and retained their phenotype, migration ability and albumin uptake functions. Human renal cell in three-dimensional culture-formed tubules, which stained positively for proximal, distal tubule and collecting duct markers, and this was confirmed by western blot. Polarity of the tubular cells was determined by the presence of E-cadherin, N-cadherin and Na-K ATPase. Colocalization of labeled albumin and proximal tubule markers proved functionality and specificity of the newly formed tubules. An in vivo study showed that cells survived in the kidney for up to 6 weeks. These findings demonstrate that human renal cell grown in three-dimensional culture are able to generate kidney structures in vitro. This system may ultimately be developed into an efficient cell-based therapy for patients with CKD.
Original Articles
In vitro reconstitution of human kidney structures for renal cell therapy
Nadia K. Guimaraes-Souza1,2,
4
, Liliya M. Yamaleyeva1, Tamer AbouShwareb1,3, Anthony Atala1,3 and
James J. Yoo1,3
1
Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston Salem, NC, USA,
2
Research
Institute Albert Einstein Jewish Hospital, Sao Paulo, Brazil and
3
Department of Urology, Wake Forest University Health Sciences,
Winston Salem, NC, USA
Correspondence and offprint requests to: James J. Yoo; E-mail: jyoo@wfubmc.edu
4
Present address: Department of Nephrology, Albert Einstein Jewish Hospital Sao Paulo, Sao Paulo, Brazil.
Abstract
Background. Recent advances in cell therapies have pro-
vided potential opportunities for the treatment of chronic
kidney diseases (CKDs). We investigated whether human
kidney structures could be preformed in vitro for sub-
sequent implantation in vivo to maximize tissue-forming
efciency.
Methods. Human renal cells were isolated from unused
donor kidneys. Human renal cells were cultured and ex-
panded. Migration was analyzed using growth factors. To
form structures, cells were placed in a three-dimensional
culture system. Cells were characterized by immunouor-
escence, western blots and uorescence-activated cell
sorting using renal cell-specic markers for podocin,
proximal and distal tubules and collecting ducts. An
albumin uptake assay was used to analyze function.
Three-dimensional cultures were implanted into athymic
rat kidneys to evaluate survival.
Results. Human renal cells were effectively expanded in
culture and retained their phenotype, migration ability and
albumin uptake functions. Human renal cell in three-di-
mensional culture-formed tubules, which stained posi-
tively for proximal, distal tubule and collecting duct
markers, and this was conrmed by western blot. Polarity
of the tubular cells was determined by the presence of E-
cadherin, N-cadherin and Na-K ATPase. Colocalization of
labeled albumin and proximal tubule markers proved
functionality and specicity of the newly formed tubules.
An in vivo study showed that cells survived in the kidney
for up to 6 weeks.
Conclusions. These ndings demonstrate that human
renal cell grown in three-dimensional culture are able to
generate kidney structures in vitro. This system may ulti-
mately be developed into an efcient cell-based therapy
for patients with CKD.
Keywords: cell therapy; chronic kidney disease; human renal cells;
three-dimensional cell culture
Introduction
Chronic kidney disease (CKD) has been steadily increasing
in the last decade. The National Kidney Foundation esti-
mates that the number of CKD patients will double in the
next 10 years [1]. The total Medicare expenditures on
patients that progress to end-stage renal disease (ESRD)
exceed $17 billion in the USA alone [2,3]. While dialysis is
the most common form of therapy used in patients with
ESRD, this modality is only able to prolong survival by par-
tially replacing the ltration function of the kidney. Renal
transplantation is the only denitive treatment that can
restore complete renal function [4]. However, increasing
demand and donor shortage make this treatment challenging.
Recent advances in cell-based therapies have provided
potential opportunities to alleviate the current challenges
of donor shortage. Many investigative studies have evalu-
ated cell therapy as an alternative treatment modality for
acute kidney disease and CKD [5,6]. Bone marrow me-
senchymal stem cells were shown to protect renal function
and prevent tissue damage after ischemia reperfusion in
animal models of acute renal failure [79]. In animal
models of chronic kidney failure, cell therapy with me-
senchymal stem cells prevented disease progression [10,
11]. Stem cells derived from human amniotic uid have
demonstrated protective effects in a mouse model of acute
tubular necrosis [12]. In addition, organ-specic stem
cells and progenitor cells have been described in the
kidney [13,14]. Multipotent progenitor cells have been
isolated and characterized from the Bowmans capsule of
adult human kidney. When these cells were injected into
acute renal failure-induced severe combined immunode-
cient mice, signs of improvement in renal function were
observed [15]. In another study, embryonic stem cells
were seeded in a decellularized rat kidney. These cells in-
teracted with the kidney scaffold and proliferated within
the glomerular, vascular and tubular structures, leading to
a loss of embryonic cellular characteristics and expression
of the markers of differentiated kidney cells [16].
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We previously demonstrated that single renal cells can
be isolated from donor tissue, expanded in vitro and rein-
troduced in vivo for renal tissue regeneration. The poten-
tial of renal cell therapy was demonstrated in a study in
which culture expanded cells were seeded onto an arti-
cial renal device and implanted in vivo. This resulted in
the formation of renal structures that produced urine-like
uid [17]. In this study, single renal cells showed the
ability to reconstitute into renal tubules and glomeruli on
the articial renal device. While single renal cells show
evidence of reconstitution into functional kidney struc-
tures, the efciency of the process of structural assembly
could not be assessed in vivo. A strategy that involves the
reconstitution of renal structures during the culture expan-
sion stage was proposed. As such, a three-dimensional
culture system that allows for reconstituting single renal
cells into kidney structures was developed which provided
a controlled platform for renal tissue formation [18]. In
this study, we investigated whether human kidney struc-
tures could be preformed in vitro for subsequent implan-
tation in vivo to maximize tissue-forming efciency.
Materials and methods
Preparation of cells
Discarded human kidneys were obtained from Carolina Donor Services
after ethical approval by the university institutional review board.
Briey, pieces of the kidney (cortex and medulla) were minced in Krebs
buffer until a gel-like consistency was obtained. Liberase blendzyme 3
(Roche, Indianapolis, IN) was added to the tissue. This solution was l-
tered in a 100-μmlter and centrifuged. The supernatant was re-sus-
pended in complete medium and plated in 15-cm culture dished plates,
as described previously [19,20]. Five different donors were tested. One
of the ve kidneys had been rejected from the donation pool due to the
conrmed diagnosis of arteriosclerosis and glomerulosclerosis.
The medium used for renal cell culture contained one part high
glucose Dulbeccos Modied Eagles Medium (Gibco Invitrogen, Carls-
bad, CA) supplemented with 10% fetal bovine serum (FBS; Gibco Invi-
trogen) and 1% penicillinstreptomycin (Gibco Invitrogen) and one part
keratinocyte serum-free medium supplemented (Gibco Invitrogen) with
2.5% FBS, 0.02% insulin, transferin and sodium selenite (ITS; Sigma
Aldrich, St Louis, MO) and 1% penicillinstreptomycin.
Medium was changed every 2 days. Cells were allowed to grow to
conuency and then 0.05% trypsin (Gibco Invitrogen) was used to
detach cells. After centrifugation, cells were plated at 1:3 ratio.
Characterization of cells
Immunouorescence and western blot analyses were performed with
different antibodies for cell characterization both in two- and three-di-
mensional cultures. Fluorescence-activated cell sorting (FACS) was per-
formed for cells in two-dimensional culture.
Immunouorescence
Cells were xed with 4% paraformaldehyde (Polysciences, Inc., Wa-
shington, PA) at room temperature for 10 min. To stain cytoplasmic pro-
teins, the cells were permeabilized with 0.1% Triton (Sigma, St Louis,
MO). Primary antibodies were added and incubated for 1 h at room
temperature. Secondary antibodies were kept in for 30 min. Control
samples for auto uorescence or non-specicuorescence were per-
formed on xed cells or tissue without the primary antibodies. Tissues
were parafn embedded; cut in 4-μm sections and xed in 10% neutral-
ized buffered formalin. Peroxidase quenching and antigen retrieval were
performed prior to staining.
Western blot
Western blot analysis was performed on cell lysates from kidney tissue.
Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel
electrophoresis, transferred to polyvinylidene uoride (PVDF)
membranes (Bio-Rad, Hercules, CA) and reacted with primary anti-
bodies [neprilysin (NEP) 1:100; erythropoietin (EPO) 1:200; Tamm
Horsfall protein (THP) 1:100; podocin 1:25]. Membranes were then in-
cubated with the appropriate horseradish peroxidase-conjugated second-
ary antibody, followed by enhanced chemiluminescence detection to
visualize bands. THP, EPO and podocin were purchased from Santa
Cruz Biotechnology; Santa Cruz, CA. NEP was purchased from Chemi-
conMillipore.
FA C S
Primary renal cells at Passage 3 were trypsinized, xed in 2% parafor-
maldehyde, permeabilized in 0.1% saponin/phosphate-buffered saline
(PBS) buffer and blocked for 30 min in permeabilization buffer contain-
ing 3% normal goat serum. Following washes in cold PBS, the cells
were re-suspended in blocking buffer at 1.0 ×10
6
cells/mL and incubated
with cell-specic unconjugated antibodies to NEP for proximal tubules
(sc-19993), epithelial membrane antigen for distal tubules (ab28081;
Abcam), THP for distal tubules and collecting ducts (sc-20631), EPO for
renal interstitial broblasts (sc-1310) and podocin for podocytes (sc-
21009). Control cells were labeled with either mouse or rabbit isotype
control antibodies (sc-2025 or sc-2027, respectively). All primary anti-
bodies were added at a concentration of 1 μg/mL and were incubated for
30 min at room temperature. After washing 3× with PBS, cells were in-
cubated with Alexa-Fluor 488-conjugated goat anti-rabbit or mouse sec-
ondary antibody (Invitrogen) for 15 min at room temperature. Cells were
washed 3× with PBS and xed with 2% paraformaldehyde. A FACSCali-
bur (Becton-Dickinson, Franklin Lakes, NJ) was used to perform ow
cytometry and results were analyzed with FlowJo software (Treestar Inc).
Functional assays
Cell migration. Cell migration assays were performed using the ECM
510 kit from Chemicon. Briey, 1.5 × 10
4
cells at Passage 3 were kept
overnight in serum-free medium, followed by incubation with either
human recombinant hepatocyte growth factor (HGF) in different concen-
trations (5, 10, 60 and 100 ng/mL); 10
7
M/mL aldosterone, 10% FBS;
epidermal growth factor (EGF) in different concentrations (1.25, 2.5 and
5 ng/mL); 0.7% ITS and combinations of 1.25 ng/mL EGF + 60 ng/mL
HGF; 2.5 ng/mL EGF + 60 ng/mL HGF + 10
7
M/mL aldosterone and 5
ng/mL EGF + 60 ng/mL HGF + 10
7
M/mL aldosterone as chemoattrac-
tants. After 4 h, the reaction was stopped and monitored uorometrically
with a FLUOstar OPTIMA microplate reader with excitation at 500 nm
and emission at 534 nm. All drugs were purchased from Sigma
Albumin uptake. Conuent two dimensional cultures at Passage 3 and
three-dimensional cultures were incubated with rhodamine-labeled
human albumin (25 µg/mL) (Sigma) at 37°C for 30 min, extensively
rinsed (3×) with PBS, xed for 30 min with 2% paraformaldehyde and
subsequently stained for NEP or epithelial membrane antigen (EMA).
Viral transfection. Lentiviral transfection was performed using the
system devised by Kages and colleagues [21]. In this system, three plas-
mids were co-transfected into a 293T cell line (ATCC, Houston, TX),
resulting in transient expression of virus packaging elements and gener-
ation of replication-incompetent Lentiviral vector particles harboring
green uorescence protein (GFP). The virus was added to human renal
cells culture and after 24 h, 50% of cells were GFP positive. After 48 h,
100% were GFP positive. Cells were passaged at a ratio of 1:3 and
allowed to grow to conuency to conrm the transfection rate.
Three-dimensional cultures. Rat tail collagen Type I (BD Falcon, San
Jose, CA), and 199× Media (Gibco Invitrogen) were mixed in a ratio of
9:1 and the pH was adjusted to 7.4 using 1× NaOH (Sigma). Cells at
Passage 3 were suspended at a concentration of 12 × 10
6
cells/mL and
added to the collagen as previously described [22]. The cellmatrix sus-
pension at a volume of 150 µL was placed in 48-well tissue culture
plates. The plate was kept in a 37°C incubator at 5% CO
2
for 20 min
allowing collagen solidication to allow a gelatin-like consistency for the
scaffolds. After solidication, medium was added to each well and
changed every 2 days for the 10-day culture period.
In vivo study. Experiments were performed using adult athymic male
rats (Harlan Laboratories). Animals were used according to protocols ap-
proved by the Wake Forest University Institutional Animal and Care and
Use Committee. Under isourane anesthesia, animals underwent a ne-
phrotomy in the right kidney and the three-dimensional culture (collagen
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scaffold with cells) was implanted within the kidney parenchyma. Suture
was performed in order to keep the scaffold in the kidney parenchyma.
The same procedure was performed in the left kidney with implantation
of a collagen scaffold without cells. Animals were euthanized at three
different time points: 1, 3 and 6 weeks post-implant.
Results
Cell culture
Cells from discarded human kidneys (age range, 2 months
to 69 years) with no known morbidities were harvested as a
heterogenous population using the same methods described
by our group for rodent renal cell harvest [18,19]. All pro-
cedures were approved by the Institutional Review Board of
Wake Forest University. Under phase contrast microscopy,
different cell morphologies (data not shown) were visualized
over multiple passages. Growth curves were analyzed from
ve different donors, and these showed that in 3 days, the
cells doubled their number. Cells were cultivated on average
for 50.8 ± 15.8 days and the population doubling in this
period was 18.52 ± 1.66 times. In 45 days, independently of
the donorsage or kidney morphology, the cell population
doubled 18 times, showing the in vitro ability of expansion
of human kidney cells (Figure 1).
Characterization of two-dimensional cell cultures
In order to determine which cell types were grown in the
heterogenous culture, immunouorescence and western
blot analyses were performed. Markers for proximal
tubule (anti-NEP), distal tubule (EMA), collecting duct
(THP), podocytes (podocinPODO) and EPO-producing
broblasts (EPO) were tested for the rst three passages.
Immunouorescence labeling showed specic staining in
all passages. Cells expressing the same markers tended to
cluster in groups distinct from other cell types in the
culture plate (Figure 2).
In agreement with the immunouorescence, the western
blot analysis was positive for the same proteins
(Figure 3). Western blot analysis using EPO antibody
showed a double band at 35 kDa in all passages. Human
kidney tissue and Knrk (normal rat kidney cells lysate)
were used as positive controls. Western blots for NEP,
THP and podocin (86, 50 and 53 kDA, respectively)
showed positive results in all test passages and the corre-
sponding positive controls. Together, the data show that
human kidney cells harvested as a heterogenous popu-
lation kept their phenotype in two-dimensional cultures
over three passages.
To determine the number of specic cells present in
Passage 3, FACS was performed. EPO-positive cells were
the most prevalent ones (54%). The second most preva-
lent was podocin (36.6%). Other markers studied in-
cluded: THP (33.2%), EMA (23%) and NEP 2.53%.
These ndings conrm the existence of subtypes of cells
in the primary culture.
Migration assay
A migration assay was performed to determine if this hetero-
genous cell population, at Passage 3, maintained the ability
to migrate toward growth factor signals. The assay showed
that 30% of cells migrate within 4 h toward all the growth
factors tested. However, no signicant differences among
the different growth factors and dosages were found.
Functional assay
To evaluate the function of cells in vitro, human recombi-
nant albumin labeled with rhodamine was added to cells
Fig. 1. Growth curve of human renal cells. Cells from different age donors
were counted after achieving conuency. Donors from different age had the
same behavior in culture. Furthermore, note that the 60-year-old donor has
the same growth curve as the other donors. This donor had 2.5 mg/dL of
creatinine and glomerulosclerosis in the kidney biopsy. In the gure, h
means human and the subsequent numbers indicate the age of the donor.
Fig. 2. Characterization of human renal cells by immunocytochemistry.
Cells from different donors in Passages 1, 2 and 3 were stained.
Polyclonal anti-EPO antibody in Passage 1 (P1) (A), EPO antibody in
Passage 2 (P2) (B), EPO antibody in Passage 3 (P3) (C), anti-NEP
antibody against proximal tubule present in Passages 1 (D), 2 (E) and 3
(F); THP antibody in Passages 1 (G), 2 (H) and 3 (I) and podocin
antibody in the Passages 1 (J), 2 (K) and 3 (L). EMA antibody in
Passage 1 (M), 2 (N) and 3 (O). Original magnication, ×20.
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growing in a monolayer on a 0.4-μm membrane. Albumin
was added to the apical side of cells and after 30 min, the
labeled albumin was visualized in the cytoplasm of some
cells (Figure 4A). Labeled albumin was not visualized in
the basolateral side of the insert. Staining for NEP (prox-
imal tubule) and EMA (distal tubule) showed albumin
uptake for the proximal tubule cells but not for the distal
tubule cells (Figure 4B and C, respectively).
Three-dimensional cell culture
Three-dimensional culturing methods were used to allow
epithelial renal cells to undergo tubulogenesis. After 10
days in the three-dimensional culture, tubule-like struc-
tures were formed. Hematoxylin and eosin staining
showed cystic structures, some showing a small degree of
extension resembling branching morphogenesis.
To obtain enhanced visualization of these structures,
immunouorescence for the different tubule markers was
analyzed with confocal microscopy. Confocal images
showed rounded and elongated structures with internal
lumen. Lentivirus GFP-transfected cells showed elongated
structures with lumen, tubule-like structures and also
some cystic structures (Figure 5). These structures in the
three-dimensional collagen culture stained positive for
markers of proximal tubule (NEP), distal tubule (EMA),
collecting duct (TammHorsfall) and EPO. Cells forming
a single structure were only positive for a single tubule
marker. Proximal and distal cells were never observed to-
gether in the same structure. Podocin antibody was not
positive in any of the three-dimensional culture
experiments.
Western blot analyses conrmed the results of the im-
munostaining. EPO, NEP and THP were positive in the
three-dimensional culture. E-cadherin, N-cadherin and
Na/K ATPase pumps were all located at the proper
locations in the cells. These results indicate that during
the tubulogenesis process in the three-dimensional
culture, epithelial tubular cells migrated and formed
tubule-like structures with adequate polarization. These
data suggest that three-dimensional culture is an excellent
tool for studying tubulogenesis, as it not only induces for-
mation of tubule-like structures but also maintains the cell
phenotypes (Figure 6).
Functionality assays
Human recombinant albumin labeled with rodhamine was
added to the three-dimensional cultures. After 30 min,
albumin was taken up by the cells (Figure 7). To identify
the type of cells incorporating albumin, immunostains
with anti-NEP and anti-EMA were performed. Proximal
tubular cells were the only ones that uptook the albumin
(Figure 7B). Thus, along with phenotype and the ability
to migrate and form tubule-like structures, these cells and
newly formed tubules kept the specialized function of
protein uptake.
In vivo study
Lentivirus GFP-transfected cells were allowed to grow
into collagen scaffolds. Scaffolds with or without cells
were surgically implanted in athymic rats. Animals
were followed for 1, 3 and 6 weeks. At these time
points, animals were euthanized under anesthesia and
Fig. 3. Panel (A) characterization of human renal cells by western blot. EPO, NEP, THP and podocin proteins expressed in Passages 1, 2 and 3
localized in the same position as the control bands. Panel (B) FACS showing EPO, NEP, THP, EMA and podocin.
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kidneys were harvested. We analyzed the survival and
integration of these human cells in vivo. After 1 week,
cells were found and located close to the implantation
site. Moreover, some tubule-like structures could be
seen in the tissue. After the rst week time point, some
cells migrated out of the collagen and integrated into
the glomerular and in the interstitial space near veins
and arteries. No tumorogenicity was observed in the
experiments. Human von Willebrand factor positive
cells were also seen (Figure 8).
Discussion
The concept of cell-based approaches has been proposed
as a method to improve, restore or replace renal function
[23,24]. In this study, we demonstrate that human renal
cells can be expanded in vitro while maintaining their
phenotype and function. In a three-dimensional culture
system, these cells generate kidney structures. Human
renal cells demonstrate the capability of cyst formation
with internal lumen as well as formation of tubule-like
Fig. 4. Human recombinant rhodamine-labeled albumin assay. (A) Overlap of phase microscopy image with uorescent rhodamine light, some cells
exhibit uorescent staining (red) in the cytoplasm corresponding to the labeled albumin, (B) anti-EMA antibody staining (green) showing that distal
tubule cells did not take up the albumin, cells with cytoplasmatic rhodamine uorescence (shown in red) were not positive for EMA, (C) proximal
tubule cells stained for NEP (green), (D) rhodamine-labeled albumin present in the cytoplasm and (E) merged picture showing albumin stain inside
the proximal renal tubular cells (red for albumin, green for anti-NEP antibody and nuclei in blue). Original magnication, ×40.
Fig. 5. Three-dimensional culturing of human renal cells. (A) Hematoxylin and eosin staining in a magnication of ×20, arrows indicate the cystic
extensions, (B) higher magnication of a cystic formation, (C) confocal image in the same magnication showing NEP stain as well as EPO
Antibody stain and (D) confocal images of GFP lentivirus-transfected cells after 10 days in the three-dimensional cultures.
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structures with proper polarization and selective uptake of
albumin. When exposed to an in vivo environment, the
cells survived and migrated to glomerular and interstitial
areas. This in vitro study may be a model for future
studies of molecular mechanisms of human kidney epi-
thelial cells.
In this study, a primary heterogeneous population of
cells harvested from discarded human kidneys was ex-
panded for up to 18 population doublings in a two-dimen-
sional culture. These cells were characterized for
expression of markers of renal differentiated epithelial
cells such as NEP, THP and EMA using immunostaining,
western blot assay and FACS up to the third passage.
Markers suggesting the presence of podocytes and EPO-
producing cells were also present in all tested cells,
suggesting the presence and expansion of those types of
cells as well. These data suggest that two-dimensional
culture can be used for expanding human renal cells and,
more interestingly, that their phenotype is maintained
throughout this process.
The in vitrostudy was conducted on ve different
kidneys from donors of different ages and genders. Of the
ve donors, a 60-year-old female had a serum creatinine
level over 2.5 mg/dL at hospital admission. This patients
kidney was discarded from the donation pool due to a
conrmed diagnosis of glomerulosclerosis. Nevertheless,
the cells derived from this kidney were able to grow and
maintain phenotype and function similar to those obtained
Fig. 6. Characterization of tubule-like structures. (A) Panel with immunouorescence and immunohistochemistry for EPO, NEP and THP at a
magnication of ×20, (B) western blot for the expression of the same proteins in the three-dimensional cultures, supernatant and pellet fractions of
the tissues were tested (Knrk: control kidney cells, HK: human kidneys, EPO, NEP, THP), (C) E-cadherin exhibited the proximal tubule tight
junctions, (D) N-cadherin demonstrated tight junctions on the basolateral membrane of distal tubule and (E) Na/K ATPase pump in the newly formed
structures.
Fig. 7. Tubule-like structures and function in the three-dimensional culture. (A) Newly formed tubule positive for EMA staining did not uptake
albumin and (B) proximal tubule cells stained for NEP exhibit cytoplasmatic inclusions of albumin.
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from other donors. Therefore, we may assume that ex-
panding renal autologous cells from CKD patients may be
a feasible approach.
Cell migration plays a basic role in many physiological
and pathophysiological processes such as embryogenesis,
immune defense, wound healing and metastasis. This
process requires the directed reorganization of the cytos-
keleton [25] the activity of ion channels and transporters
[2630] and coordinated formation of focal adhesion con-
tacts to the extracellular matrix [31]. Results from our
migration assay showed that human renal cells expanded
in culture kept their ability to coordinate all this machin-
ery and migrate toward different growth factors such as
HGF, EGF and aldosterone, which are the same growth
factors that coordinate the kidney embryogenesis [3235].
Epithelial cells grown in monolayers are frequently
used as a model for epithelial polarity [36,37]. Using this
methodology in combination with the use of human re-
combinant rhodamine-labeled albumin, we demonstrated
that human renal cells are able to uptake albumin. Proxi-
mal tubular cells marked with anti-NEP took up this
albumin, while distal tubular cells labeled with anti-EMA
were exposed to albumin and did not uptake the protein
[38].
Once the phenotype and migration capability were de-
monstrated in two-dimensional culture, these cells were
cultured in a collagen Type I matrix, with addition of
growth factors such as HGF, EGF and ITS. Ten days in
this culture system allowed cells to form rounded struc-
tures with an internal lumen, tight junctions and
Fig. 8. In vivostudy. Athymic rat kidney implanted with cells seeded in three-dimensional collagen scaffolds. (A) 1-week time point showing cells
stained with anti-GFP antibody adjacent to the collagen. Arrows show rat kidney cells that are negative for GFP. Original magnication ×20, (B) ×63
magnication showing tubule-like structure positive for anti-GFP in the interface between scaffold and normal parenchyma (arrows show cells
negative for GFP), (C) 3-week time point showing human renal cells stained for anti-GFP migrating from the collagen toward the tissue (black
arrows indicate the GFP-positive cells and red arrows indicate negative cells), (D) human cells present in glomerular structure. Data from the 6-week
time point were similar to that seen at the 3-week time point, and they are not shown here. (E) Human-specic von Willebrand-positive staining in
the rat parenchyma.
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extensions similar to the ones in embryonic development
and also tubule-like structures. These experiments were
performed with cells from all donors, and the same nd-
ings were seen among all of them. Therefore, we may
presume that independently of age and gender, human
renal cells are potentially able to form cysts and tubule-
like structures when exposed to a favorable environment.
Epithelial morphogenesis is a highly complex multistep
process that requires coordinated cell-to-cell and cell-to-
extracellular matrix interactions as well as time to create
three-dimensional structures [3941]. In this study, this
technology was used in human cells from different ages
to examine the ability of the cells to undergo tubule for-
mation. Cells from different tubular segments were able to
group together and form polarized functional tubules in
vitro. In addition, these three-dimensional culture systems
may provide an exceptional in vitro model for the study
of cell signaling and cellmatrix interactions.
In the three-dimensional system, cell-to-cell and cell-
matrix interactions allowed the formation of tubule-like
structures that expressed tubular epithelial markers in im-
munostains and western blot assays. These cells could
form differentiated tubule-like structures. However, it was
quite clear that proximal and distal tubular cells did not
coexist in the same structure, emphasizing their ability to
identify each other. Podocytes were not able to survive in
this environment. This result was not unexpected since
this particular type of cell presents a low proliferation rate
and demands special culture conditions [42].
Albuminuria is an important indicator and possible
pathogenic factor in CKD progression. Reabsorption of
albumin from the glomerular ltrate is made via receptor-
mediated endocytosis in the proximal tubule in the
normal kidney [43]. In this three-dimensional culture
system, tubule-like structures formed by human renal cells
selectively absorbed albumin. Distal cells stained for
EMA did not take up albumin. Meanwhile, single cells
and tubule-like structures expressing NEP growing in the
collagen exhibited rhodamine-labeled albumin in their
cytoplasm. This structure formation system has great
potential for therapeutic uses particularly when combined
with collagen since collagen is Food and Drug Adminis-
tration (FDA) approved.
In order to evaluate the potential cell therapy use of
these in vitro grown cells, the three-dimensional cul-
tured cells were implanted into athymic rat kidneys. To
identify these human cells inside the rat kidney, cells
were transfected with GFP lentivirus and further stained
for GFP. Cells transfected with GFP lentivirus cultured
in three-dimensional conditions formed the same struc-
tures. These scaffolds were implanted in athymic rats as
a controlled environment for tracking these human
cells. After 1 week, the collagen scaffold could be
easily visualized and cells were stained with an anti-
GFP antibody. Tubule-like structures and single cells
were identied in the collagen area. In the interface
between collagen and normal parenchyma, human cells
could also be visualized. After 3 weeks, these cells
were out of the scaffold and integrated either in the
glomerular or in the interstitial areas. These results
were quite promising showing that human renal cells
implanted in vivo were able to migrate and integrate
into existing renal structures.
In conclusion, human renal cells can be harvested from
normal and diseased tissue and grown in culture while
maintaining phenotype and function. When exposed to
extracellular matrix, these cells formed tubule-like struc-
tures with proper polarization and function. Collagen scaf-
folds implanted in vivo allowed cells to survive and
migrate toward the parenchyma and integrate into glomer-
ular and interstitial structures. Therefore, this model could
be an important tool for studies in the physiology of
human renal cells with probable implications in future
clinical therapies.
Acknowledgements. The authors thank Drs Jennifer Olson and John
Jackson for editorial assistance and Kathryn Stern and Kenneth Gyabaah
for technical assistance. This work was supported, in part, by Tengion,
Inc. through a sponsored research agreement.
Transparency declaration. The authors would like to disclose that
Drs J.J.Y. and A.A. are consultants for Tengion, Inc.
Conict of interest statement. None declared.
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    • "Ectopic organogenesis has been extended to several sites in vivo, since the kidney capsule restricts space availability for the maturating renal primordia. Transplantation of E15 rat metanephroi into omentum of non-immunosuppressed adult rats resulted in a normal kidney structure and function following uretero-uterostomy, surviving as long as 32 weeks (Hammerman, 2002). A recent study by the Lagasse group has reported the successful organogenesis of the kidney using the mouse lymph node as a developmental niche. "
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    • "Recent studies by our group have demonstrated that primary renal cells isolated from human kidneys facilitate beneficial effects toward the recovery of renal functions. As previously described, we developed a cell isolation and culture system to obtain sufficient numbers of human primary renal cells for cell therapy [12]. We have established two rodent kidney injury models by varying the length of the renal ischemic time [83]. "
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