JNEPHROL 2009; 22: 312-317
THOROUGH CRITICAL APPRAISALS
www.sin-italy.org/jnonline – www.jnephrol.com
Xenobiotic kidney organogenesis: a new
avenue for renal transplantation
Takashi Yokoo1,2, Tetsuya Kawamura2
Abstract
Currently many efforts are being made to apply regenerative medicine to clinical renal diseases. It has
been suggested that some renal diseases which maintain renal structure can be treated by infusion of stem
cells isolated from the bone marrow or adult kidney.
However such cell-based therapy cannot be applied
to the treatment of chronic renal disease, in which renal structure, including the kidney scaffold, is totally
disrupted. Therefore, absolute kidney regeneration is
needed to rebuild a whole functional kidney de novo
and eliminate the requirement for dialysis. However,
due to the anatomical complexity of the kidney and the
need for communication between each cell to fulfill renal function, the kidney has been labeled as the most
difficult organ to regenerate. Only a small number of
groups are investigating the potential for reconstructing an organized and functional kidney structure, and,
among them, we are using the developing xenoembryo
as an organ factory for this purpose. Here we review
the challenges faced in developing a whole functional
kidney de novo and discuss the obstacles which must
be overcome before clinical use is possible.
Key words: Embryo, Kidney regeneration, Mesenchymal stem cell, Metanephros, Xenobiology
Introduction
The kidney retains the potential to regenerate itself as long
as the damage is not too severe and the kidney structure remains intact. Therefore, regenerative medicine for
such kidney diseases should aim to activate or support
Project Laboratory for Kidney Regeneration, Institute of
DNA Medicine, Tokyo - Japan
2
Division of Nephrology and Hypertension, Department
of Internal Medicine, Jikei University School of Medicine,
Tokyo - Japan
1
this potential. However, in cases of irreversible damage to
the kidney, as can occur with long-term dialysis, the selfrenewal function is totally lost. Thus, any application of
regenerative medicine in end-stage renal disease (ESRD)
will require the development of an entire functional kidney
de novo, as a whole organ.
ESRD is the terminal stage of chronic renal failure which
needs to be considered in regenerative medicine. Most patients with ESRD entering dialysis programs have type 2 diabetes, chronic glomerulonephritis or hypertension (1). The
number of ESRD patients requiring dialysis has increased
markedly worldwide, mainly due to the significantly extended acceptance criteria for dialysis, which now include more
elderly and diabetic patients, as well as those with other
severe comorbidities (2). Current trends in maintenance
dialysis population dynamics show the estimated annual
worldwide cost of maintenance ESRD therapy at close to
US $75 billion. The size of this global maintenance dialysis
population is expanding at a rate of 7% per year (3). If this
current trend continues, the dialysis population will exceed
2 million patients by the year 2010, and the aggregate cost
will be more than US $1 trillion over the coming decade
(3). This will render dialysis impractical in the near future
as a therapeutic choice for ESRD patients. Although kidneys may be transplanted successfully, the lack of suitable
transplantable organs has prevented kidney transplantation from becoming a practical solution for most cases
of ESRD. Thus, there is a need for a new type of therapy
where patients with ESRD may discontinue dialysis, and, in
this regard, kidney regeneration has considerable potential.
However, the kidney is anatomically complicated and resident cells must communicate with each other to function.
Therefore, a regenerated whole therapeutic kidney must
contain fully organized and orchestrated cells able to fulfill
their function. Thus, unlike treatment of acute renal failure,
regenerative medicine for ESRD requires a novel approach
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to build a functional whole kidney de novo.
The present article reviews such challenges and discusses
the obstacles which must be overcome before clinical use
of any novel therapies can be contemplated.
Previous trials to establish a whole
kidney de novo
Previous trials can be divided into several groups depending
on the source of regenerated kidney. One of the possible
sources for this purpose is the embryonic kidney (metanephros). A metanephros transplanted into the renal cortex
of a host mouse may continue to grow, and the developed
metanephros contains vascularized glomeruli and mature
proximal tubules and may have the capacity for glomerular
filtration (4). Dekel et al (5) also reported that metanephroi
from porcine embryos implanted under the kidney capsule
of immunodeficient mice can differentiate into a functional
nephron. Surprisingly, the concentration of urea nitrogen and
creatinine was higher in the cyst fluid arising from this transplant than in the sera of the transplanted mice, suggesting
that the transplant was functioning to filter the host blood
and produce urine, which was the first demonstration of urine
production from an artificial kidney (5). A metanephros can
also be transplanted into a host omentum, where it is not
confined by a tight kidney capsule. These transplants can
assume a kidney-like shape in situ that is approximately
one third the diameter of the native kidney (6). It has been
shown that in the case of xenotransplantation (pig metanephros to rat omentum), immunosuppressants were
required because, without these agents, the transplants
disappeared soon after transplantation. Interestingly, the
graft pig metanephros was slightly larger in volume (diameter and weight) than a normal rat kidney. Furthermore, the
transplanted tissue produced urine, and surprisingly, after
intact ureteroureterostomy with the ureter of the removed
kidney, anephric rats started to void urine and showed a
prolonged lifespan (7). These experiments were based on
previous studies showing minimal immunogenicity in tissues harvested at earlier gestational stages, including the
metanephros (8), and the results provide the rationale for
the usefulness of the metanephros from early embryos as
a potential source of transplantable regenerated kidney to
address the shortage of organs for kidney transplantation.
Recently, Osafune et al (9) reported that a select population from metanephric mesenchyme is enough to form a
whole kidney. They showed that a single SAL-like 1 highlyexpressing cell from the metanephric mesenchyme forms
a 3-dimensional kidney structure consisting of glomeruli
and renal tubules (9). This system is useful for examining
the mechanisms of renal progenitor differentiation, but also
suggests the possibility of establishing a whole kidney from
a single stem cell from metanephric mesenchyme.
Although the ethical issues remain to be resolved, derivatives from fertilized ova may be another source for the
artificial kidney. Xenopus presumptive ectoderm, which
becomes epidermis and neural tissue in normal development, contains pluripotent stem cells which can be differentiated into multilineage tissue cells under particular
culture conditions (10). Chan and colleagues (11) designed conditions for the induction of pronephric tubulelike structures from animal caps that involved a combination of activin and retinoic acid for only 3 hours. This
pronephros-like tissue was transplanted into bilaterally
nephrectomized tadpoles to test for functional integrity as
a pronephros. Bilateral pronephrectomy induces severe
edema in tadpoles owing to its inability to excrete internal water, and tadpoles die within 9 days; transplantation
of the pronephros-like unit at least partially corrected the
edema and tadpoles survived for up to 1 month. To our
knowledge, this is the only study to establish a transplantable functional whole kidney unit in vitro.
Embryonic stem (ES) cells are undifferentiated pluripotent
stem cells isolated from the inner cell mass of blastocysts
(12) and have the capacity to differentiate into several cell
types of mesodermal, endodermal and ectodermal lineage, depending on culture conditions. Therefore they are
assumed to be a potential source of cells for tissue regeneration. Although there are no published reports describing
an ES cell–derived whole functional kidney, several groups
have shown that ES cells can differentiate into renal structures if they are injected into immunosuppressed mice (13,
14) or transplanted into developing metanephroi cultured
in vitro (15, 16). In addition, after a single injection into
developing live newborn mice kidneys, ES cells expressing brachyury, stably integrate into proximal tubules with
normal morphology and polarization for 7 months without teratoma formation (16). These data highlight ES cells
as a potential source of renal stem cells for regenerative
therapy; however they are non-self cells and may evoke
an immunoresponse if the resultant organ is transplanted
without any manipulation.
To overcome such concerns, Lanza et al (17) attempted to
establish a self-kidney unit to eliminate the problem of the
immune response. To generate a histocompatible kidney for
artificial organ transplantation, they used a nuclear transplantation technique, in which dermal fibroblasts isolated from an
adult cow were transferred into enucleated bovine oocytes
and transferred nonsurgically into progestin-synchronized
recipients. A renal device, seeded with cloned metanephric
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cells was then transplanted into the cow, from which dermal
fibroblasts were isolated. Surprisingly it appeared to produce
a urine-like liquid, suggesting the use of nuclear transplantation for renal regeneration is possible without the risks and
long-term effects of immunosuppression.
Based on these accumulating challenges, the ideal features
of a regenerated artificial kidney for ESRD can be defined:
i.e., it must have a precise kidney structure, produce urine
and grow with no or a minimum requirement for immunosuppression. With these features in mind, we attempted to
derived cells were scattered throughout the rudimentary
metanephros and were morphologically identical to tubular
epithelial cells, interstitial cells and glomerular epithelial cells
(19), demonstrating that using a xenobiotic developmental
process for growing embryos allows endogenous hMSCs
to undergo an epithelial conversion and be transformed into
an orchestrated nephron consisting of glomerular epithelial
cells (podocytes) linked to tubular epithelial cells (19).
Urine production from the regenerated kidney
establish the ideal artificial kidney.
Xenoembryo as an organ factory
Rebuilding the kidney structure
The first step was to try and reconstruct an organized and
functional kidney structure using a developing heterozoic
embryo as an “organ factory.” During embryogenesis, a
single fertilized cell develops into a whole body within 10
months in humans and 20 days in rodents. This neonate
has every organ positioned correctly, indicating that a single fertilized ovum contains a blueprint from which the body,
including the kidney, can be built. Therefore, we sought to
“borrow” this programming of a developing embryo by implanting stem cells at the site of organogenesis.
During development of the metanephros, glial cell–derived
neurotrophic factor (GDNF) is initially expressed in the metanephric mesenchyme to initiate development (18). Therefore, we hypothesized that GDNF-expressing mesenchymal
stem cells (MSCs) may differentiate into kidney structures if
positioned at the budding site and stimulated by numerous
factors spatially and temporally identical to those found in
the developmental milieu.
We first established a culture system combining a whole embryo culture system, followed by a metanephric organ culture.
This relay culture system allowed the development of the
metanephros from structures present before budding until
the occurrence of complete organogenesis ex utero. In this
system, embryos were isolated from the mother before budding and were grown in a culture bottle until the formation of
a rudimentary kidney so that it could be further developed by
organ culture in vitro (19). Using this combination, rudimentary
kidneys continued to grow in vitro, indicating that the metanephros can complete development ex utero even if the embryo
is dissected prior to sprouting of the ureteric bud.
Based on these results, GDNF-expressing human MSCs
(hMSCs) were microinjected at the site of budding and
subjected to relay culture. After the relay culture, hMSCs-
To acquire the ability to produce urine, the regenerated kidney must have the vascular system of the recipient; therefore, the primary system was modified to allow for vascular
integration from the recipient to form a functional nephron.
We used the previously described findings of Rogers and
colleagues (6), who found that the metanephros can grow
and differentiate into a functional renal unit with integration
of recipient blood vessels if it is implanted into the omentum.
Because we found that only metanephroi from rat embryos
older than embryonic day 13.5 (E13.5) developed successfully, the relay culture system was modified so that organ
culture was terminated within 24 hours, by which time the
metanephros was sufficiently developed, and the kidney primordia could be transplanted into the omentum (termed the
modified relay culture system). As a result, a hMSC-derived
neo-kidney was generated that was equivalent to a human
nephron (20). A flow diagram of the establishment of neokidney is shown in Figure 1. Using the LacZ transgenic rat
as a recipient (21) and electron microscopic analysis, we
proved that the vasculature of the neo-kidney in the omentum originated from the host and communicated with the
host circulation, suggesting its viability to collect and filter
the host blood to produce urine (20). Indeed, the neo-kidney
left in the omentum for another 2 weeks developed hydronephrosis, confirming the ability of the neo-kidney to produce
urine (if the ureter was buried under the fat of the omentum,
the urine would have no egress, resulting in hydronephrosis). Analysis of the liquid from the expanded ureter showed
higher levels of urea nitrogen and creatinine than in the recipient sera, but similar to the native urine (20).
Physiologically regulated secretion of
erythropoietin from the neo-kidney
To show whether the neo-kidney produced by our system
could fulfill other renal functions as well as urine production, erythropoietin (EPO) production was examined, since
the production of EPO to maintain erythropoiesis is another
important function of the kidney (22). We found that the
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Fig. 1 - A flow diagram of the establishment of neo-kidney. hMSCs = human mesenchymal stem cells.
Fig. 2 - Which path would you like to take? Currently most patients with end-stage renal disease have an arteriovenous (A-V)
fistula created and start lifelong dialysis (red arrows). Instead, if our system is achieved, isolated mesenchymal stem cells
(MSCs) from their bone marrow are cultured in growing embryos for a given time to develop into kidney tissue, followed by
autologous implantation into the omentum of the same patient. The kidney primordia eventually become a self-organ that
produces the patient’s urine. The patient is then hopefully free from dialysis and without renal disease.
neo-kidney can produce human EPO, which is stimulated
by induction of anemia. We also found that the levels of
EPO generated by the neo-kidney, in response to anemia
in rats in which native EPO was suppressed, were sufficient
to restore red cell recovery to a rate similar to that in control
rats (23). These data suggest that this system preserves
the normal physiological regulation of EPO levels, and the
neo-kidney derived from hMSCs may be able to fulfill all
renal functions, including urine production.
Perspective and conclusion
Recent advances in stem cell research have brought
the possibility of organ regeneration using somatic stem
cells for clinical organ replacement one step closer to
realization. However, anatomically complicated organs,
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such as the kidney and liver, have proven more refractory
to stem cell–based regenerative techniques.
In this article we have described the current challenges for
whole kidney regeneration from autologous stem cells. We
have succeeded in establishing a system by which bone
marrow–derived MSCs can develop into a kidney unit which
can fulfill renal functions including urine production in the
rat omentum. However, it should be noted that our current
system is still in the developmental phase and a long way
from being established for clinical use.
For example, our current system cannot exchange the
Wolffian duct for one of human origin, as the collecting duct
to the ureter consists of host embryo tissue. Although the
developing renal anlagen are less immunogenic than the
developed kidney, even a small contamination of chimeric
cells might evoke an unexpectedly large immunoreaction.
Therefore, we are currently attempting to overcome this
hurdle by 2 different approaches: (a) eliminating xenogenic
cells before transplant into the omentum using a transgenic
host that carries a regulated suicide gene and (b) exchanging the posterior part of the Wolffian duct for human during
its elongation so that the collecting duct in the neo-kidney
may be of host origin. In addition, the resultant size of the
neo-kidney produced by the current system is too small
for human renal function, even though the neo-kidney does
not need to be the same size as the native kidney for relief
from dialysis. We need to seek larger host embryos to establish larger organs more suited for use in humans. It has
recently been reported that pig metanephroi transplanted
into rat omentum may develop a larger volume (diameter
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Financial support: This work was supported by a grant from the
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Japan and by the Uehara Memorial Foundation.
Conflict of interest statement: None declared.
Address for correspondence:
Takashi Yokoo, MD, PhD
Department of Internal Medicine
The Jikei University School of Medicine
3-25-8 Nishi-Shimbashi, Minato-ku,
105-8461 Tokyo, Japan
tyokoo@jikei.ac.jp
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Received: January 19, 2008
Revised: March 04, 2008
Accepted: July 08, 2008
© Società Italiana di Nefrologia
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