RENAL ONTOGENY AND EVALUATION OF DECELLULARIZED KIDNEY
SCAFFOLDS FOR TISSUE ENGINEERING IN A RHESUS MONKEY MODEL
A Project
Presented to the faculty of the Department of Biological Sciences
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
Biological Sciences
(Stem Cell)
by
Jennifer Leigh Keyser
SPRING
2012
RENAL ONTOGENY AND EVALUATION OF DECELLULARIZED KIDNEY
SCAFFOLDS FOR TISSUE ENGINEERING IN A RHESUS MONKEY MODEL
A Project
by
Jennifer Leigh Keyser
Approved by:
__________________________________, Committee Chair
Thomas Landerholm
__________________________________, Second Reader
Jan Nolta
__________________________________, Third Reader
Christine Kirvan
___________________
Date
ii
Student: Jennifer Leigh Keyser
I certify that this student has met the requirements for format contained in the University
format manual, and that this project is suitable for shelving in the Library and credit is to
be awarded for the project.
__________________________, Graduate Coordinator
Ronald Coleman
Department of Biological Sciences
iii
___________________
Date
Abstract
of
RENAL ONTOGENY AND EVALUATION OF DECELLULARIZED KIDNEY
SCAFFOLDS FOR TISSUE ENGINEERING IN A RHESUS MONKEY MODEL
by
Jennifer Leigh Keyser
The prevalence of end stage renal disease (ESRD) is dramatically increasing. It is
estimated that the number of people in the United States undergoing treatment for chronic
kidney disease will rise from 500,000 to two million by the year 2030. In infants and
children, ESRD is commonly caused by developmental abnormalities including
obstruction of the fetal urinary tract.
Characterization of developing glomerular
precursors including renal vesicles, C- and S-shaped bodies, and podocytes is necessary
in order to determine effective ways to recapitulate ontogeny with the goal of repair.
New strategies to repair damaged organs using decellularized biological scaffolds are
being developed, with the hope of using cell therapy for tissue regeneration within a
natural framework.
Decellularized scaffolds must serve as a support for cellular
interactions and not result in an immune response. This study characterizes fetal kidney
development in a clinically relevant rhesus monkey model and assesses whether MHC
Class I or Class II proteins remain in a decellularized kidney scaffold.
This was
addressed by staining fetal kidneys across gestation with hematoxylin and eosin (H&E),
iv
and performing immunohistochemistry (IHC) and enzyme-linked immunosorbant assay
(ELISA) on fresh kidneys and kidneys decellularized in 1% sodium dodecyl sulfate
(SDS). Results show that renal development in the rhesus monkey mimics that in the
human. Glomerular precursors are abundant in the cortex during the first and second
trimesters, and by the third trimester, nearly all precursors have differentiated to mature
glomeruli. Near term kidneys from the fetal rhesus monkey model of obstructive renal
dysplasia show cystic glomeruli with cystic dilatation of Bowman’s space, and abnormal
glomerular tuft development when compared with unobstructed kidneys. The podocyte
marker, WT-1, is strongly expressed in the condensed mesenchyme and nephrogenic
zone at the cortical border during the first and second trimesters. By the third trimester,
WT-1 expression is confined to mature glomeruli. IHC and ELISA revealed that HLA-E
and HLA-DR were removed from the scaffolds during decellularization, while modest
amounts of HLA-DQ remained in juvenile decellularized kidney scaffolds. The results
from this study add to the growing base of research concerning the use of decellularized
scaffolds and will contribute to the ability to repair or replace damaged kidneys in vivo.
_______________________, Committee Chair
Thomas Landerholm
_______________________
Date
v
ACKNOWLEDGEMENTS
I would like to thank Drs. Tom Peavy, Tom Landerholm, and Hao Nguyen for the
patience and support they have given, and to Dr. Alice Tarantal for opening her lab to me
and guiding my master's research. This work was supported and made possible by the
California Institute for Regenerative Medicine. The members of the Tarantal laboratory
deserve a special thanks for lending a helping hand whenever one was needed, especially
doctoral student, Karina Nakayama, who has acted a mentor throughout this project. I
owe my heartfelt gratitude to Robyn Bilski, for supporting me through all stages of this
endeavor. Last but certainly not least, I would like to thank my parents, Penny Keyser
and the late Randy Keyser, who always pushed me to be the best I could be.
vi
TABLE OF CONTENTS
Page
Acknowledgments........................................................................................................ vi
List of Figures ........................................................................................................... viii
INTRODUCTION ........................................................................................................ 1
MATERIALS AND METHODS ................................................................................ 14
Animals ................................................................................................................ 14
Hematoxylin and Eosin (H&E) Staining ............................................................. 14
Decellularization and Immunohistochemistry (IHC) .......................................... 15
Enzyme Linked Immunosorbent Assay (ELISA) ................................................ 16
Statistical Analysis............................................................................................... 17
RESULTS ................................................................................................................... 18
DISCUSSION ............................................................................................................. 24
Literature Cited ........................................................................................................... 28
vii
LIST OF FIGURES
Figures
Page
1. Stages of development ............................................................................................ 5
2. This cross section of a mature glomerulus shows the relationship between the
different cell types and the glomerular basement membrane (GBM .......................6
3. H&E staining of fetal rhesus monkey kidneys across gestation ........................... 19
4. Immunohistochemical staining for WT-1 ............................................................. 20
5. Immunohistochemical staining for MHC Class I (HLA-E) and MHC Class II
(HLA-DR and HLA-DQ) proteins in fresh and decellularized kidneys ............... 22
6. ELISA of MHC Class II proteins after decellularization...................................... 23
viii
1
INTRODUCTION
Animal models are invaluable for obtaining information about human biological
processes and disease. Rodents have proven highly useful but have some limitations for
translation due to the many physiologic differences when compared to human and
nonhuman primates [Bontrop, 2001; Goodman and Check, 2002; Shively and Clarkson,
2009]. For example, rodents lack receptors for many immunotherapeutics designed to
enhance or suppress immune response [Buse et al., 2003], and there are considerable
differences in the innate and adaptive immune response in rodents and primates [Mestas
and Hughes, 2004]. Mice have a short lifespan and are challenging to use to study the
long-term effects of treatment such as cell or organ engraftment, whereas nonhuman
primates excel in this role [Trobridge and Kiem, 2010]. Nonhuman primates have proven
to be an important translational model for human development and disease due to
similarities in reproduction and gestational length, patterns of organogenesis, and growth
characteristics [Lee et al., 2001; Tarantal, 2005]. The strong similarities with humans in
spatial and temporal patterns of development make nonhuman primates prime candidates
for the study of kidney development and disease [Batchelder et al., 2009, 2010; Matsell
and Tarantal, 2002], and new therapies for treatment [Matsell et al., 2002; Tarantal et al.,
2001, 2012; Tarantal and Nakayama, 2011].
The prevalence of end stage renal disease (ESRD) is dramatically increasing.
Currently, over 500,000 people in the United States are undergoing treatment for chronic
2
kidney disease, and it is estimated that this number will rise to two million by the year
2030 [Coresh et al., 2005; Stevens et al., 2006]. ESRD in infants and children is
commonly caused by developmental abnormalities. According to the North American
Pediatric Renal Trials and Collaborative Studies Annual Report in 2011, obstructive
uropathy (blockage of urine flow) and renal dysplasia account for as many as 15% of
diagnoses in pediatric kidney transplant recipients.
Obstructive nephropathy (urine
backup into the kidneys due to blockage) is associated with renal dysplasia (abnormal
kidney development), hydronephrosis (kidney swelling), and altered nephron endowment
(the number of functioning nephrons) and glomerular development [Chevalier et al.,
2010; Matsell et al., 2002]. Despite the prevalence and poor prognosis of fetal urinary
tract obstruction, little progress has been made in understanding the causes, or in
developing effective methods of treatment [Tarantal et al., 2001]. Obstructive uropathy
is just one of many renal diseases in need of new treatments. Kidney dialysis or whole
organ transplantation remains the only option for patients who progress to ESRD.
Kidney transplantation is the best option, but there exists a severe shortage of kidneys.
According to the Organ Procurement and Transplantation Network, as of March 2012,
there are more than 91,000 people waiting for a kidney transplant, and less than 20% of
those on the waiting list will receive transplants annually. Dialysis is the other option for
ESRD patients; however, dialysis does not completely replace kidney function and limits
the quality of life of the patient. The inequity between the prevalence of ESRD and
viable treatments supports that an alternative to whole organ transplantation and dialysis
3
is needed. Stem and progenitor cell therapy for repair or regeneration may prove to be a
solution to this problem.
Tarantal et al. [2001] developed a fetal rhesus monkey model of obstructive renal
dysplasia that reproduces the characteristic features of obstructive renal disease in
humans. Loss of podocytes, which leads to decreased glomerular filtration, was observed
in fetal monkey dysplastic kidneys as early as the S-shaped stage of nephron
development. Gross changes in glomerular morphology were also observed in this model
[Butt et al., 2007; Hiatt et al., 2010; Matsell et al., 2002; Tarantal et al., 2001]. These
changes suggest that the precursor glomerular structures (renal vesicles and C- and Sshaped bodies) necessary for normal development may be able to be replaced or repaired.
In order to fully appreciate the pathology associated with obstructive
nephropathy, one must have a full understanding of normal renal development. This is
essential in order to appreciate normal processes and determine effective ways to
recapitulate ontogeny with the goal of repair. Kidney development in the rhesus monkey
(Macaca mulatta) follows a pattern similar to that of humans [Batchelder et al., 2009;
2010]. The kidneys develop from the intermediate mesoderm and differentiate first
towards a temporary kidney, the mesonephros, and finally to the definitive or permanent
kidney, the metanephros. There are two components critical to the normal formation of
the definitive kidney, the ureteric bud and the blastema. The ureteric bud forms the
future collecting system of the kidney and the blastema is the region of formation of the
4
glomeruli. Molecular interactions between these two structures are critical for normal
kidneys to form. If this interaction does not occur, the kidney will not form (renal
agenesis). The ureteric bud, important in the formation of the collecting system, emerges
from the nephric duct (the primitive drainage duct), and invades the metanephric
mesenchyme and undergoes branching. Figure 1 [Dressler, 2006] shows the stages of
normal kidney development. Mesenchymal cells form aggregates at the tips of the
branching ureteric bud, forming the blastema. These cell aggregates begin the process of
mesenchyme-to-epithelial conversion.
Renal vesicles form from the differentiating
epithelium; a cleft forms in the renal vesicle, creating the characteristic C-shaped body,
followed by the formation of another cleft, producing the S-shaped body. The distal end
of the S-shaped body fuses to the adjacent ureteric bud, creating an epithelial tubule
which leads to the collecting duct. The glomerulus (basic unit of filtration) develops at
the proximal end of the tubule. During the S-shaped stage of development, endothelial
cells invade the cleft, and it has been suggested that mesangial cells (which provide
structure, elasticity, and regulate plasma flow) may be generated from these endothelial
cells. Epithelial cells proximal to the invading endothelium differentiate to visceral
epithelial cells (podocytes) while cells in the distal region of the S- shaped body
differentiate to parietal epithelial cells.
Figure 2 [Dressler, 2006] shows the cell types in the mature glomerulus. Mature
glomeruli are composed of podocytes, parietal epithelial cells, capillary endothelial cells,
5
Figure 1. Stages of nephron development. Mesenchymal aggregates form at the ureteric
bud tips and undergo mesenchyme-to-epithelial transition (MET) to form the renal
vesicle, which differentiates to the C- and S-shaped bodies and ultimately leads to
formation of a glomerulus [Dressler, 2006].
6
Figure 2. Cell types of the glomerulus. This cross section of a mature glomerulus shows
the relationship between the different cell types and the glomerular basement membrane
(GBM). Of particular importance are the podocytes, which act as a filtration barrier; this
podocyte barrier is shown on the right in an electron micrograph [Dressler, 2006].
7
and mesangial cells [Dressler, 2006; 2009; Saxon, 1987; Vize et al., 2003]. Each of these
cell types play a role in the kidney and must be replaced if damaged by disease. In terms
of species differences, nephrogenesis in the mouse continues postnatally whereas
nephrogenesis is completed prenatally in human and nonhuman primates [Batchelder et
al., 2010].
The disruption of function or loss of podocytes is common in renal disease
[Pavenstädt et al., 2003], and is observed in the fetal rhesus monkey model of obstructive
renal dysplasia [Matsell et al. 2002].
Podocytes are terminally differentiated cells
surrounding the glomerular basement membrane [Pavenstädt et al., 2003]. These cells
interact with the glomerular capillaries and adjacent podocytes and are important in
protein retention by acting as a size and charge barrier, and regulating glomerular
pressure [Quaggin et al., 2008; Shankland, 2006]. Podocytes are a key cell type in
glomerular filtration, and are unable to effectively regenerate in order to replenish lost
podocytes [Durvasula and Shankland, 2006]. The identification of renal precursor cells
and ability to induce stem/progenitor cells to differentiate into podocytes could provide a
potential treatment for many renal diseases involving podocyte damage or loss [Leapley
et al., 2009].
Extensive research has focused on the search for and characterization of renal
progenitor cells [Challen et al., 2004; Little, 2006; Nishinakamura, 2008], but a
progenitor cell population has not yet been identified, and many of the molecular
8
processes that drive kidney development remain elusive.
Understanding precursor
structures associated with nephrogenesis will aid in developing a timeline of
differentiation that may advance the search for a progenitor cell population and promote
further elucidation of the role(s) of specific molecules in kidney development which can
be used for future stem cell therapy studies.
A complete understanding of kidney
ontogeny is needed to fully appreciate normal development and ways to recapitulate this
process in a model of kidney disease.
Using established renal developmental markers, Leapley et al. [2009] defined
criteria for assessing the developmental stage of renal cortical cells in culture; Nestin was
used to identify podocyte progenitors and Synaptopodin was used to identify mature
podocytes; potentiating the ability to characterize progression and maturation of renal
cells in vitro. In a more recent study, Batchelder et al. [2010] characterized the patterns
of expression of renal developmental markers during ontogeny of the rhesus monkey,
allowing for a more complete understanding of development and nephrogenesis. These
data are essential in tissue engineering and recapitulating kidney development and
maturation. Studies by Leapley et al. [2009] have demonstrated the capability to culture
podocytes and showed that renal extracellular matrix (ECM) supports podocyte
progenitor proliferation and maintenance in vitro. Other recent studies by this group
supports the hypothesis that decellularized renal scaffolds containing native ECM which
may serve as a template for repair of kidneys damaged by disease [Nakayama et al.,
2010; 2011].
9
Further research is underway to develop new strategies to repair damaged organs
using decellularized biological scaffolds, with the hope of using cell therapy for tissue
regeneration within a natural framework. This is important because it is unlikely that cell
transplantation in kidneys damaged by disease without a scaffold will be effective.
Current thought suggests that decellularized organ scaffolds with intact ECM may be the
most effective material due to the remaining organ-specific structure and ECM proteins
that facilitate cell-cell and cell-ECM interactions that are essential for organogenesis
[Crapo et al., 2011; Gilbert et al. 2006; Song and Ott, 2011]. To date, advances in the use
of decellularized tissue have been utilized for the heart [Ott et al., 2008], trachea
[Baiguera et al., 2010; Conconi et al., 2005], liver [Uygun et al., 2010], and lung [Ott, et
al., 2010; Petersen et al., 2010).
Many challenges remain to be overcome if decellularized scaffolds are to be
considered for use in vivo, either as whole organs or as templates for transplant of
organized structures for cells. One goal with cell transplantation is to avoid the need for
immunosuppression which is necessary if the donor cells are not from the host
(autologous) or from a matched donor [Chinen and Buckley, 2010; Fuchs et al., 2001].
This is also important for decellularized/recellularized scaffolds because remnants of
components from the donor could result in an immune response and rejection posttransplantation [Gilbert et al., 2006]. For the potential use of decellularized scaffolds to
be achieved, the scaffold must serve as a support for cellular interactions and not result in
an immune response.
10
Nakayama et al. [2011] demonstrated the ability of decellularized renal scaffolds
from different age groups of monkey donors to support cell migration and infiltration
using a kidney explant/scaffold in vitro model, as well as to maintain expression of renal
developmental markers when seeded with fetal monkey renal tubular and glomerular
fractions. Choosing the best cell population(s) for recellularizing decellularized renal
scaffolds is essential if the goal is to recapitulate development and form functional kidney
structures. A renal stem/progenitor cell capable of differentiating to all of the cell types
of the kidney would likely be the optimal population, but to date, no such population has
been identified [Harari-Steinberg et al., 2011]. Due to the large number of cell types
found in the kidney, the use of multipotent or pluripotent cells for repopulation is highly
desirable. For example, the use of embryonic stem (ESCs) or induced pluripotent stem
(iPS) cells present unique potential. These cells hold great promise for kidney tissue
engineering which can only be unlocked by providing the precise molecular signaling
required to drive the cells towards defined renal lineages and create the multitude of cell
populations needed to repair the kidney.
It has also been shown that endothelial
progenitor cells have angiogenic potential and could provide additional support to
facilitate vascularization when used with pluripotent cells such as ES or iPS cells [Dekel
et al., 2006; Sangidorj et al., 2010].
Determining what remains in a scaffold after decellularization is important not
only for assessing the presence of potential immunogenic proteins or molecules, but also
to confirm the presence of desired ECM components. Fibronectin and laminin (adhesion
11
proteins), collagen, glycosaminoglycans (GAGs), elastin fibers, and growth factors may
all promote cell adhesion and infiltration and their retention is important in scaffold
decellularization [Gilbert et al., 2006].
Nakayama et al. [2010] demonstrated the
preservation of collagen types I and IV, heparan sulfate proteoglycan (GAG), fibronectin,
and laminin in renal scaffolds using 1% sodium dodecyl sulfate (SDS) for
decellularization.
Although it is currently unclear if residual DNA may prove
problematic, eliminating donor DNA may have benefits in reducing the potential risk of
immunogenicity [Gilbert et al., 2009; Lotze et al., 2007].
There are many proteins and cellular components that must be eliminated from a
decellularized scaffold to avoid the potential for host immune response.
Major
histocompatibility complex (MHC) proteins represent an important class of proteins
capable of triggering graft rejection. There are two main types of MHC molecules, MHC
Class I and MHC Class II. MHC Class I molecules are expressed by all nucleated cells,
while MHC Class II molecules are expressed only by activated T-cells, B-cells,
macrophages, dendritic cells, and epithelial cells of the thymus. A component of this
study was focused on assessing whether a subset of MHC molecules are indeed removed
using the decellularization protocol developed by Nakayama et al. [2010]. Intact, as well
as denatured MHC proteins can be recognized by the host immune system. Host antigen
presenting cells (APCs) are capable of engulfing native or processed MHC molecules and
presenting them for allorecognition by T lymphocytes (T-cells), in turn triggering an
immune response and rejection of the graft.
12
MHC molecules have been proposed to be the most important alloantigens for
triggering rejection by allorecognition [Wood and Goto, 2011]. Unmatched donor MHC
molecules can be recognized by direct, indirect, or semi-direct allorecognition. Direct
allorecognition occurs when intact MHC-molecules (complexed with a peptide) are
presented by donor APCs and recognized by host T-cells. Similarly, in semi-direct
allorecognition, donor MHC molecules interact with T-cells, however, in this pathway,
the complexes are first captured and presented by host APCs. Indirect allorecognition is
accomplished when donor MHC molecules are degraded and the resulting peptides are
presented by host APCs [Caballero et al., 2006; Heeger, 2003; Wood and Goto, 2011].
Studies by Nakayama et al. [2010; 2011] have clearly shown all cells are removed
from the scaffold during the decellularization protocol, thereby eliminating the direct
allorecognition pathway; however, the presence of any solubilized donor MHC molecules
could lead to graft rejection. Still, it is unlikely that the semi-direct pathway could be
utilized by the host immune system, because the decellularization protocol uses a
denaturing detergent, thus decreasing the likelihood that intact, functional MHC
molecules remain. Denatured MHC molecules have the potential for allorecognition by
the indirect pathway; therefore it is essential to ensure that degraded fragments of MHC
molecules are eliminated from the scaffold.
The objectives of this study were to: (1) evaluate the ontogeny of the rhesus
monkey kidney by studying morphological changes and developmental time points with
13
the ultimate goal of effectively recellularizing decellularized kidney scaffolds, and (2)
assess whether MHC Class I or Class II proteins remain in a decellularized kidney
scaffold.
14
MATERIALS AND METHODS
Animals
All protocols followed the requirements of the Animal Welfare Act and were
implemented after approval from the Institutional Animal Care and Use Committee at the
University of California, Davis. Adult female rhesus monkeys were bred and identified
as pregnant with ultrasound and fetal growth monitored sonographically during gestation
[Tarantal, 2005]. Gestation in the rhesus monkey is 165 ± 10 days, and is divided into
trimesters by 55-day increments [Tarantal and Gargosky 1995]. Fetal rhesus monkey
kidneys from the first trimester (50 days gestation), second trimester (80 and 100 days
gestation), and third trimester (120 and 150 days gestation) (N=15) were collected
according to established methods [Tarantal et al., 2001]. For comparison, juvenile
kidneys (N=6) were also collected and immediately placed in RPMI media (Life
Technologies, Grand Island, NY) until processing. All experiments were performed
under aseptic conditions.
Hematoxylin and Eosin (H&E) Staining
Fetal kidneys (N=15) were harvested and fixed in 10% phosphate buffered
formalin, then embedded in paraffin. Embedded tissues were sectioned (5 µm) and
mounted on glass slides. Tissues were deparaffinized in xylene and rehydrated in a
graded series of ethanol [Batchelder et al., 2009]. The tissues were stained for 90
seconds in Harris Hematoxylin (Thermo Fisher Scientific, Waltham, MA) and 10 seconds
15
in eosin Y solution (Thermo Fisher Scientific). Following staining, the tissues were
dehydrated in ethanol followed by xylene. The slides were mounted using Cytoseal
(Thermo Fisher Scientific) and a coverslip applied.
Decellularization and Immunohistochemisty (IHC)
Transverse kidney sections were placed either in 1% SDS decellularization (Life
Technologies) solution at 4°C, according to established protocols [Nakayama et al.,
2010], or fixed in 10% phosphate buffered formalin. For ELISA (see below), biopsies
were taken from each section using an 8 mm biopsy punch (Med Plus, Monsey, NY),
then placed in 1% SDS decellularization solution at 4°C or flash frozen in liquid nitrogen
and stored at ≤-80°C until use. Decellularization solution was changed after 8 hours, and
then every 72 hours until the tissues were transparent (12-15 days) [Nakayama et al.,
2010]. Decellularized kidney scaffolds were washed in phosphate buffered saline (PBS)
overnight. The scaffolds were transferred to 70% ethanol for sterilization, rehydrated in
PBS overnight, then fixed in 10% phosphate buffered formalin for IHC, or stored in PBS
and 1% penicillin-streptomycin (Life Technologies) for ELISA. Fresh and decellularized
kidneys for IHC were then embedded in paraffin. Fresh and decellularized paraffinembedded kidneys were sectioned (5 µm) and H&E staining was performed to assess
morphology.
IHC was performed using the Envision + System-HRP kit (Dako,
Carpinteria, CA).
Slides were deparaffinized in xylene, then rehydrated in graded
concentrations of ethanol. Slides were incubated with peroxidase blocker for 5 minutes,
followed by two washes in PBS. Heat-mediated antigen retrieval in Nuclear Decloaker
16
(pH 9, Biocare Medical, Concord, CA) was performed, followed by washes in PBS.
Primary antibodies were diluted in primary antibody buffer (5% bovine serum albumin
[BSA], 0.1% fish skin gel) were incubated with slides for 30 minutes at room
temperature. Primary antibodies against MHC Class I were HLA-E (clone MEM-E02;
Thermo Fisher Scientific) and for MHC Class II proteins were HLA-DR (clone LN-3;
Invitrogen, Grand Island, NY), and HLA-DQ (clone 4B7-E2; Abcam, Cambridge, MA).
HLA-E was diluted 1:100, HLA-DR was diluted 1:50, and HLA-DQ diluted 1:100. The
primary antibody used to identify podocytes, Wilms’ Tumor Protein (WT-1) (clone 6FH2; Millipore, Billerica, MA) was diluted 1:200 to 1:1000 (optimized for each
specimen). Immunoglobulin matched isotype controls were utilized for each antibody.
Slides were washed twice in PBS, then incubated with horseradish peroxidase (HRP)
labeled polymer conjugated to anti-mouse secondary antibody (Dako) for 30 minutes.
After PBS, the HRP substrate, 3,3’ Diaminobenzidine (DAB), was applied for 5 minutes,
followed by thorough washing in distilled water.
Slides were counterstained with
hematoxylin, dehydrated in graded ethanol followed by xylene, mounted with Cytoseal
(Thermo Fisher Scientific) permanent mounting medium, and a coverslip applied.
Enzyme-linked Immunosorbent Assay (ELISA)
Tissue Extraction Reagent I (Life Technologies) and Protease Inhibitor Cocktail
(Sigma-Aldrich, St. Louis, MO) were added to flash frozen and decellularized kidney
biopsies at 1 ml per 100 mg of tissue. The biopsies were thoroughly ground using a
disposable mortar and pestle. Ground tissues were homogenized for 3 minutes using the
17
Roche MagNA Lyser (Roche, Indianapolis, IN), followed by centrifugation for 5 minutes
at 10,000 RPM to pellet debris. Supernatant was transferred to a clean tube and stored on
ice for immediate use. Samples were diluted in 0.05 M carbonate-bicarbonate coating
buffer (Thermo Fisher Scientific) and applied to a 96-well ELISA plate in triplicate.
Plates were incubated overnight at 4°C. Wells were washed twice with 0.05% Tween-20
(Sigma-Aldrich) in PBS.
Non-specific binding was blocked with 5% BSA Fraction V
(Life Technologies) in PBS for 1 hour. After washing, each well was incubated with
mouse anti-HLA-DR or anti-HLA-DQ antibody diluted 1:5000 for 2 hours, followed by
washing and incubation with the secondary antibody goat anti-mouse IgG conjugated to
HRP (Dako) for 1 hour. Matched isotype controls were utilized for each sample. The
plates were washed and the HRP substrate 3,3,5,5-Tetramethylbenzidine (TMB) was
applied. A 0.5 M H2SO4 stop solution was applied and the absorbance read using the
BioRad Microplate Reader Model 680 in conjunction with BioRad Microplate Manager
Software, version 5.2.1 (Bio-Rad Laboratories, Hercules, CA).
Statistical Analysis
All data are shown as mean ± standard error of the mean. Statistical analysis was
performed using a two tailed Student’s t-test and data were considered statistically
significant at p <0.05.
18
RESULTS
Renal ontogeny in the rhesus monkey was assessed across gestation using H&E
staining and IHC for WT-1 to assess podocytes. Results confirmed that the presence of
developing structures in the monkey kidney is similar to the human [Saxen, 1987;
Dressler, 2006] and highlights the pathologic changes in the monkey model of
obstructive renal disease (Figure 3).
During the first and second trimesters, renal vesicles are actively differentiating
into C-shaped bodies and S-shaped bodies. By the third trimester, glomerular precursors
have reached the more mature stages consisting of S-shaped bodies and mature
glomeruli, and near term, all precursors have fully differentiated to mature glomeruli.
Near term, kidneys from the fetal rhesus monkey model of obstructive renal dysplasia
typically show cystic glomeruli with cystic dilatation of Bowman’s space and abnormal
glomerular tuft development when compared with unobstructed kidneys, including
dilated glomeruli with podocyte loss [Butt et al., 2007; Tarantal et al., 2001].
WT-1, the marker expressed by podocytes during all stages of development was
stained to assess the presence and localization of podocytes. During normal ontogeny,
WT-1 is strongly expressed in the condensed mesenchyme of the cortex and in
glomerular precursors during the first and second trimesters and is progressively confined
to glomeruli during the third trimester (Figure 4).
19
Figure 3. H&E staining of fetal rhesus monkey kidneys across gestation. Glomerular
precursors: renal vesicles (RV), C-shaped bodies (CB), and S-shaped bodies (SB) are
abundant in the cortex during the first and second trimesters. By the third trimester,
primarily mature glomeruli (G) are present in the cortex. Near term (150 days gestation),
kidneys from the fetal rhesus monkey model of obstructive renal dysplasia show cystic
glomeruli (cg) with cystic dilatation of Bowman’s space (bs), and abnormal glomerular
tuft (gt) development when compared with unobstructed kidneys.
20
Figure 4. Immunohistochemical staining for WT-1. WT-1 is strongly expressed in the
condensed mesenchyme (CM) and nephrogenic zone at the cortical border during the first
and second trimesters (CB, C-shaped body; RV, renal vesicle). By the third trimester,
WT-1 expression is confined to podocytes of mature glomeruli (G).
21
IHC was performed to assess the presence of MHC Class I and Class II proteins
in decellularized fetal and juvenile kidneys.
Fresh, control fetal kidneys showed
expression of HLA-E and HLA-DQ, but not HLA-DR. HLA-E, HLA-DQ, and HLA-DR
were all expressed in fresh juvenile kidneys. After decellularization, the two MHC
proteins observed in fetal kidneys (HLA-E and HLA-DQ) were undetectable by IHC.
HLA-E and HLA-DR were undetectable in decellularized juvenile kidney scaffolds,
while modest HLA-DQ staining within the glomerular capillaries was detected (Figure
5).
Indirect ELISA was utilized to assess the relative quantity of MHC proteins
remaining in decellularized kidney scaffolds compared to the amount present in fresh,
flash frozen kidneys (Figure 6). In the ELISA analysis of HLA-DR, there was no
statistically significant difference using the Student's t-test in the mean absorbance levels
of 1X decellularized kidney homogenates versus fresh kidney homogenates diluted
1:100,000 (0.001%). These data suggest that there was > 99.999% elimination of HLADR (p > 0.05). Any remaining HLA-DR was below the level of detection of the assay.
The analysis of HLA-DQ revealed that the mean absorbance of fresh kidney
homogenates diluted 1:10,000 (0.01%) and decellularized kidney homogenates at 1X
showed no statistically significant differences, suggesting that 0.01% of the HLA-DQ
protein present in fresh kidneys remained after the decellularization process.
22
Figure 5. Immunohistochemical staining for MHC Class I (HLA-E) and MHC Class II
(HLA-DR and HLA-DQ) proteins in fresh and decellularized kidneys. In fresh kidneys,
HLA-E and HLA-DQ are expressed in both fetal and juvenile kidneys, but HLA-DR
expression was observed only in juvenile kidneys. HLA-E and HLA-DR were not
detectable by IHC after decellularization. Modest HLA-DQ staining was detected in the
glomeruli of juvenile kidneys after decellularization.
23
A
HLA-DR Detection by ELISA
0.1400
Mean Absorbance
0.1200
*
*
0.1000
*
*
0.0800
*
*
0.0600
0.0400
0.0200
0.0000
0.1%
0.02%
0.01%
0.005%
0.002%
0.001%
1X
Kidney Homogenate Concentration
Fresh Kidney
Decellularized Kidney Scaffold
B
HLA-DQ Detection by ELISA
0.12
Mean Absorbance
0.10
*
*
*
0.08
0.06
0.04
0.02
0.00
0.1%
0.02%
0.01%
0.005%
1X
Kidney Homogenate Concentration
Figure 6. ELISA of MHC Class II proteins after decellularization. Analysis of A) HLADR and B) HLA-DQ revealed that decellularization eliminated HLA-DR and HLA-DQ
to the level present in 0.001% and 0.01% fresh kidney homogenates, respectively
(*denotes statistical significance, determined using the Student's t-test, p < 0.05).
24
DISCUSSION
Each year, thousands of Americans die waiting for a kidney transplant. Tissue
engineering and regenerative medicine hold the potential to greatly reduce the inequity
between the number of people awaiting transplants and the number of kidney donors by
creating viable treatment alternatives.
Studies have focused on obstructive renal disease, a leading cause of pediatric
kidney failure for which there are currently few treatment options.
Since the
ramifications of disease appear early in development, it may be necessary to consider
treatment prenatally. Here, it will be essential to recapitulate normal renal development
which requires a thorough understanding of all facets of renal ontogeny, including
morphological changes and developmental time points, as well as key cell populations,
molecular markers, and signaling between developing structures. The transition from
broad WT-1 expression in the nephrogenic zone to the confinement of WT-1 to
podocytes in mature glomeruli illustrates the differentiation of podocyte precursors to
mature podocytes. Batchelder et al. [2010] characterized the expression of renal markers
in the rhesus monkey kidney, and showed that the expression of WT-1 and Nestin
increased in the metanephric mesenchyme as Pax2 expression declined.
During
glomerular precursor differentiation, Pax2, Nestin, and WT-1 were expressed in podocyte
progenitors found in the cleft of the S-shaped body, and Nestin and WT-1 expression
persisted in mature podocytes, along with Synaptopodin. This study has further
25
characterized WT-1 expression across gestation and in the context of renal development,
and together, these data reveal molecular markers and timepoints that will be crucial to
recapitulate in renal tissue engineering.
A decellularized scaffold is an important natural template for kidney regeneration
because it provides the natural arrangement of matrix molecules likely to support the
extensive crosstalk between early donor cell populations, and can provide space for cells
from the recipient to integrate. This study addressed essential steps in moving towards
effective recellularization strategies by further characterizing fetal kidney development
and assessment of MHC markers likely to trigger immune responses within the
decellularized donor scaffolds that may impact the survival and effectiveness of the grafts
post-transplant.
Many obstacles must be overcome to recapitulate kidney development. The
complex development and three-dimensional structure, coupled with the multitude of cell
types that reside in the kidney, make it a particularly difficult organ to engineer. A
logical starting point is the framework on which cells can be grown to recapitulate the
interaction between key cell populations.
Decellularized kidneys can serve as the
scaffold, containing native ECM structure and proteins that may facilitate the necessary
interactions and signaling for organogenesis.
In order for these scaffolds to be
considered, it is important that any elements that may result in rejection are eliminated.
Transplantation of biological materials can trigger an innate immune response.
If
26
immunogens are present within the scaffold, the adaptive immune response will destroy
and remove the graft.
The use of decellularized scaffolds requires that multiple
components be taken into consideration; not only must all cells be removed, but care
must be taken and protocols refined to ensure that cellular debris, especially proteins
involved in allorecognition such as MHC proteins, are completely removed.
This study has shown that the decellularization protocol developed by Nakayama
et al. [2010] successfully removes all cells from the donor kidney sections, while
maintaining native ECM structures. Expression of HLA-DR, a MHC Class II protein,
was not detected in fetal kidneys which is important because it implies that the stage of
immune maturation is an advantage in the use of near term scaffolds for recellularization
and future transplantation. The expressed MHC proteins, HLA-E and HLA-DQ, were
also not detected in decellularized fetal kidney scaffolds when evaluated by IHC, thus
supporting that the process was efficient.
Two of the three analyzed MHC proteins, HLA-E and HLA-DR, were
undetectable in decellularized kidney scaffolds from juveniles. However, HLA-DQ was
modestly observed in decellularized kidney scaffolds, which was confirmed by ELISA.
Further studies will be needed to determine the most effective methods for removal of
these trace elements, as well as to focus on detection and quantification of other MHC
molecules that may be relevant such as HLA-A, HLA-B, and HLA-C (MHC Class I) and
HLA-DM, HLA-DO, and HLA-DP (MHC Class II) [Klein and Sato, 2000].
One
27
potential limiting factor may be identifying antibodies that can effectively cross-react
with rhesus monkey tissues for these other MHC molecules.
Other challenges exist for repair of damaged or diseased kidneys, including
choosing the best cell population(s) for seeding/recellularizing the scaffold, and ensuring
the cells repopulate the scaffold as desired and are functional. Results from this study
reveal the intrinsic difficulty of ensuring that all potential immunogens are removed
during decellularization with the current protocols used in these studies. Alternative
decellularization strategies such as the use of SDS perfusion in a circulating bioreactor
may prove beneficial in the complete removal of unwanted components of the scaffold.
Future investigations focus on determining the best cell type(s) for recapitulating
kidney development, the potential use of bioreactors to enhance recellularization, and the
potential for in vivo immunogenicity.
These and other recent studies illustrate the
difficulties that must still be overcome for the use of decellularized scaffolds in vivo, yet
validate the optimism of current research in the field of tissue engineering and
regenerative medicine.
28
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