Progenitor Cell Therapy for Kidney Regeneration 15

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209 End-stage renal disease (ESRD) is a deadly dis-

ease unless supportive treatment is adminis- tered in the form of hemodialysis, peritoneal dialysis, or kidney transplantation. The morbid- ity, poor quality of life, and extensive burden on the health service of patients on chronic dialysis and the shortage of kidney donors for renal transplantation are major factors that prompt the development of alternative therapies for patients with ESRD.¹

Regenerative medicine is focused on the development of cells, tissues, and organs for the purpose of restoring function through transplantation. The general thought is that replacement, repair, and restoration of function is best accomplished by cells, tissues, or organs that can perform the appropriate physiologic/metabolic duties better than any mechanical device, recombinant protein therapeutic, or chemical compound.²

Because of the kidney’s is complex structure and function, its reconstruction represents a major challenge. Thus, during the past few years, sophisticated bioengineering methods of renal cell replacement have combined biologic and synthetic components. For instance, a renal tubule assist device engineered of porcine renal proximal tubule cells and connected in series to a hemofiltration device within an extracorporeal blood circuit was shown to be beneficial in acutely uremic animals and might therefore prove to be valuable in the treatment of acute renal failure.^3,4

Induction of appropriate kidney differentiation and growth out of stem or progenitor cell

populations represents an attractive option to combat kidney disease and possibly chronic kidney donor shortage. In that regard, the use of embryonic and adult stem cells as starting material offers new and powerful strategies for future kidney tissue development and engineering.

Growing Demands for Kidney Allograft Transplantation

Organ transplantation has been one of the major medical advances of the past 30 years.

A growing number of patients can be transplanted successfully because of the continuing progress in surgery and medical treatments, and the development of new immunosuppres- sive drugs.⁵ About 1 million people have received an organ, 73 kidneys from cadaver donors have survived more than 25 years, 17 heart and 27 liver transplant recipients more than 15 years.^6,7Although the transplant community attempts to keep up with the increasing demand for transplantable organs, the supply continues to decrease far short of the need. This also applies to kidney transplant programs. It has been estimated that the number of patients with ESRD – for most of whom renal transplantation is the treatment of choice – is increasing at the rate of 7%–8% per year in the United States.⁸The United Network for Organ Sharing (UNOS) database shows that between 1988 and April 2002, the number of patients on a waiting list for renal transplantation increased from

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Progenitor Cell Therapy for Kidney Regeneration

Benjamin Dekel and Yair Reisner

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13,943 to 51,753 patients.⁹From 1988 to 2000, the patients’ waiting time has almost tripled from a median of 400 days to more than 1100 days,⁹ and the number of patients who have died every year awaiting a cadaveric renal transplantation has increased from 736 to 2875, a 290% increase.⁹Moreover, in 2000, only 26%

of the patients on the waiting list for a kidney transplant actually underwent renal transplantation.⁹Also, in most European countries, waiting lists for kidney transplantation are in a similar or worse condition than a decade ago,⁶ making the severe shortage of cadaveric organ donors the major obstacle in preventing the full development of a transplant program and imposing a severe limit to the number of patients who benefit from this form of therapy worldwide. The challenge of expanding the donor pool has forced the scientific community to explore new avenues for organ replacement.

Stem Cells

Stem cells are defined as clonogenic cells that are capable of both self-renewal and multilin- eage differentiation.¹⁰The fertilized zygote has the highest developmental potential (totipotent stem cells), because it can generate all three embryonic germ layers and all extra-embryonic tissues, including the placenta. The same property is present in embryonic stem (ES) cells from morula (4–5 days after fecundation).

Stem cells are pluripotent if they can form progenitors of all three embryonic germ layers, but not the extra-embryonic tissues. Currently, human pluripotent cells include embryonic carcinoma cells, derived from teratomas, embryonic germ cells, derived from primordial germ cells that populate the embryonic gonad, and ES cells.¹¹ ES cells are derived from the inner cell mass of the blastocyst stage embryos within the first 5–7 days after an egg is fertilized by the spermatozoon. ES cells can readily be cultured in an undifferentiated state for extended periods of time before a stimulus for differentiation is provided. Lower quantities of ES cells are present in fetal tissues (30–60 days of life). Many human ES cell lines have been derived worldwide, but only 78 cell lines met the eligibility criteria for federally funded research. A human ES cell registry is main- tained by the National Institutes of Health,

which collects the characteristics of some of these cell lines. To produce ES cell lines whose genetic material is identical to that of its source, nuclear cloning may be used. Two types of nuclear cloning, reproductive cloning and therapeutic cloning, have been described.¹² Reproductive cloning is used to generate an embryo that has the genetic material of the nucleus donor. This embryo can then be implanted into the uterus of a female to produce an infant that is a clone of the donor.

Therapeutic cloning is used to generate early- stage embryos whose genetic material is identical to that of its intended recipient, allowing derivation of ES cell lines in vitro with a potential to become almost any type of cell in the adult body. Although presently exhibiting problems typical of cloning in general, including premature aging and high rate of mortality, this approach clearly might be useful in tissue and organ replacement applications.¹² Some applications might include the treatment of neurodegenerative diseases, diabetes, and end-stage kidney diseases. The use of transplantable tissues and organs derived from therapeutic cloning may lead to the avoidance of immune responses that are typically associated with transplantation of nonautologous tissues.

Finally, somatic stem cells have been identified in several organs of both aborted fetuses (>60 days of life) and adults. They have been shown to differentiate into a limited number of cell types. For example, neural stem cells produce neurons and glial cells, and bone marrow stem cells produce all types of blood cells.^13,14However, some somatic stem cells are able to cross lineage boundaries under certain conditions. Perhaps the best example of adult stem cell plasticity is bone marrow stem cells.

Bone marrow is the source of mesenchymal stem cells, which form cells of nonhematopoi- etic system, including cardiac myocytes, hepa- tocytes, endothelial cells, and epithelial cells of the gastrointestinal tract during tissue repair,^13,14and hematopoietic stem cells, which are the ancestors of erythrocytes, granulo- cytes, monocytes, lymphocytes, and platelets.

The use of human ES cells versus adult stem cells is under rigorous ethical debate, with the current political status strongly favoring adult stem cell processes.¹¹ The plasticity of adult stem cells to transdifferentiate from one lineage pathway to another is also under careful scientific scrutiny.^15,16

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Adult Stem and Progenitor Cells for Renal Repair

Repair of Endothelium

The kidney harbors several different types of endothelial cells, in particular the endothelium of the macrovasculature, the peritubular capillary endothelium, and the glomerular endothelium.

Besides participation in the filtration, reabsorp- tion, and nutrition of the renal tissue, endothelial cells have a key role in recovery from several renal diseases. The evident question therefore is whether endothelial progenitor cells (EPCs) can also participate in regeneration and maintenance of these renal endothelial cell types. Evidence for the existence of EPCs has mostly been derived from cardiovascular research on ischemia and angiogenesis.¹⁷Circulating progenitor cells for endothelial cells were first shown by Asahara et al.¹⁸In their experiments, CD34+ leukocytes were isolated from human adult peripheral blood. These cells were subsequently cultured and differentiated into an endothelial cell-like phenotype. That these EPCs are different from circulating mature endothelial cells was elegantly shown by Lin et al.¹⁹In bone marrow transplant patients, circulating endothelial cells from both donor (bone marrow-derived) and host origin were cultured from peripheral blood. Interestingly, the bone marrow-derived cells were markedly more proliferative than the host type cells and quickly outgrew circulating mature endothelial cells. EPCs can stimulate vascular repair processes in different ways. Several authors have shown that EPCs are involved in new vessel formation by incorporating in the vessel wall.¹⁸ Furthermore, EPCs stimulate vessel formation by secreting pro-angiogenetic factors such as vascular endothelial growth factor and basic fibroblast growth factor, which subsequently enhance proliferation and migration of resident cells.²⁰

A number of studies have addressed maintenance and regeneration of the specialized renal glomerular capillaries. Pabst and Sterzel²¹ have reported that, in normal rats, the rate of total glomerular cell renewal is about 1% per day with the endothelial fraction being the most pre- dominant cell type. However, in response to injury, the rate of vascular regeneration could well be increased. An established model to study glomerular injury and repair in rats is experimental anti-Thy1.1 glomerulonephritis. Injection of a

complement-fixing antibody to the mesangial cell antigen Thy1.1 causes acute mesangiolysis and matrix dissolution, leading to ballooning of glomerular capillaries, formation of aneurysms, and loss of endothelial cells. In the subsequent repair phase, increased proliferation and migration of endothelial and mesangial cells are observed, resulting in partial restoration of glomerular structure and function. Using this model, it was shown that glomerular capillary repair is associated with a marked increase in endothelial cell proliferation.²² Several studies have provided evidence that circulating EPCs may contribute to glomerular capillary repair. Recent experiments with rat hematopoietic chimeras demonstrated low levels of bone marrow-derived cells staining for the rat endothelial cell antigen RECA-1.²³ The number of these cells gradually increased over time suggesting that EPCs contribute to normal physiologic glomerular endothelial cell turnover. After anti-Thy1.1-induced glomerular injury, a fourfold increase in bone marrow-derived endothelial cells in the glomeruli was observed.²³These data indicate that glomerular repair cannot only be attributed to migration and proliferation of resident endothelial cells but also involves bone marrow-derived cells.

Participation of circulating EPCs in renal regeneration has also been demonstrated in human adults. Williams and Alvarez²⁴were the first to report the presence of host type endothelial cells in kidney allografts. Lagaaij et al.²⁵ reported that in human renal transplants the extent of replacement of donor endothelial cells lining the peritubular capillaries by cells originating from the recipient was related to the severity of vascular injury. They suggested that this endothelial replacement could be explained by the involvement of host-derived EPCs.

Rookmaaker et al.²⁶demonstrated male, donor- derived endothelial cells in the renal macrovasculature of a female patient who developed thrombotic microangiopathy after gender-mis- matched bone marrow transplantation. Taken together, these observations confirm a novel role for bone marrow-derived endothelial cells in maintenance and repair of renal endothelium.

Repair of Mesangium

Glomerular mesangial cells provide structural capillary support to the glomerulus and display a

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pericyte- or smooth muscle cell-like phenotype.

They have a central role in the pathogenesis of a number of human and experimental glomerular inflammatory diseases. In particular, mesangial hyperplasia is a prominent histopathologic fea- ture associated with impaired glomerular function. Although transient hyperplasia is thought to reflect a physiologic response required for successful glomerular reconstitution and renal tissue repair, tight regulation of mesangial proliferation, function, and apoptosis is needed for recovery without fibrosis.

Initially, mesangial maintenance and repair after injury was thought to depend solely on proliferation of viable resident intraglomerular mesangial cells. These mature mesangial cells dedifferentiate before they proliferate, as is described by el Nahas.²⁷Similar to the glomerular endothelial cells, in normal rats, mesangial cell turnover amounts to less then 1% per day.²¹ Hugo et al.²⁸demonstrated that during recovery of anti-Thy1.1 glomerulonephritis, proliferating immature mesangial cells migrated from the juxtaglomerular apparatus and hilar region into the glomerulus.

Reminiscent to mesangial cell recruitment during embryonic glomerulogenesis, the involvement of extraglomerular mesangial progenitor cells in glomerular repair was reported by several investigators.²⁹ Imasawa et al.³⁰ demonstrated the involvement of bone marrow- derived cells in normal mesangial cell turnover.

Lethally irradiated mice transplanted with T cell-depleted bone marrow from syngeneic donors, transgenic for green fluorescent protein, manifested a time-dependent increase in green fluorescent protein-positive cells in their glomeruli. When isolated and cultured, these cells stained positive for the mesangial cell marker desmin and the cells contracted in response to angiotensin II, confirming that bone marrow-derived cells have the potential to differentiate into glomerular mesangial cells.

Similar experiments with mice transplanted with purified clonally expanded hematopoietic progenitor cells were conducted by Masuya et al.³¹ to confirm the hematopoietic origin of bone marrow-derived mesangial cells.

In similar experiments, using a rat allogenic bone marrow transplant model and antibodies to the mesangial cell-specific antigen Thy1 (ox7), Rookmaaker et al.²³confirmed this time- dependent increase of bone marrow-derived mesangial cells in the glomerulus. Both Ito

et al.³²and Rookmaaker et al.²³observed a major increase of bone marrow-derived mesangial cells during recovery from anti-Thy1.1-induced mesangiolysis in bone marrow transplantation models in rats.

Cornacchia et al.³³demonstrated that glomerulosclerosis can be transmitted by bone marrow transplantation in mice. Transplantation of bone marrow cells from sclerosis-prone mice into normal recipients with the same genetic background, invoked glomerulosclerosis in the recipients.

These data not only point to the contribution of bone marrow-derived cells to glomerular maintenance and repair but also show that dysfunctional or diseased mesangial progenitor cells can have a negative influence on the kidney.

Tubular Regeneration

The renal tubule is known for its high capacity for regeneration. Acute tubular necrosis, as a result of ischemia or toxic substances, can be followed by active migration and proliferation to restore normal tissue architecture and function.³⁴ Different sources of these proliferating progenitor cells have been reported. Humes et al.³ isolated resident proliferative epithelial cells from the tubuli of mature rabbit kidneys and demonstrated that these cells displayed a high capacity for self-renewal and differentiated into complete three-dimensional tubular structures in vitro. Similar experiments were later performed with human epithelial cells.⁴

Bone marrow-derived extrarenal tubular progenitor cells were reported by Poulsom et al.³⁵In female mouse recipients of male bone marrow grafts, colocalization of Y chromosomes and tubular epithelial cell markers was observed, suggesting participation of bone marrow- derived cells in normal tubular cell turnover.

The potential importance of the role of bone marrow-derived cells in tubular repair was demonstrated by Kale et al.³⁶When LacZ gene- positive bone marrow stem cells (Sca⁺Lin⁻ hematopoietic stem cells) were transplanted into wild-type mice, renal ischemia was associated with the occurrence of LacZ-positive (bone marrow-derived) tubular cells. It was estimated that the majority of the tubular cells after tubular repair were bone marrow derived. Moreover, bone marrow ablation diminished functional recovery after tubular ischemia, whereas infusion of a progenitor cell reversed this effect,

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suggesting an important functional role for the hematopoietic stem cell in tubular repair.

Furthermore, injection of mesenchymal stem cells of male bone marrow origin after renal injury induced in mice by the anticancer agent cisplatin remarkably protected cisplatin-treated syngeneic female mice from renal function impairment and severe tubular injury. Y chromosome-containing cells localized in the con- text of the tubular epithelial lining and displayed binding sites for Lens culinaris lectin, indicating that mesenchymal stem cells engraft the dam- aged kidney and differentiate into tubular epithelial cells, thereby restoring renal structure and function. Mesenchymal stem cells markedly accelerated tubular proliferation in response to cisplatin-induced damage, as revealed by higher numbers of Ki-67-positive cells within the tubuli with respect to cisplatin-treated mice that were given saline.³⁷Also, in humans, there are some indications for bone marrow-derived tubular repair.³⁸When male kidney transplant patients who received a female kidney and who recov- ered from acute tubular necrosis were studied, a Y chromosome could be demonstrated in few (less than 1%) of the tubular cells. Although the functional importance of this phenomenon in the human situation is still dubious, these experiments do provide a proof-of-principle observa- tion on bone marrow-derived tubular repair.

To explore the presence of renal progenitor cells that are actively engaged in tubular regeneration after injury, the existence of label-retain- ing cells (LRCs; slow-cycling cells) in normal rat kidneys by in vivo bromodeoxyuridine labeling was examined. LRCs were scattered among renal epithelial tubular cells of normal rat kidneys.

During the recovery after renal ischemia, LRCs underwent cell division and most of them became positive for proliferating cell nuclear antigen. At an early phase of tubular regeneration, descendants of LRCs expressed a mesenchymal marker, vimentin, and eventually became positive for an epithelial marker, E-cad- herin, after multiple cell divisions. These findings suggested that LRCs function as a source for regenerating and replacing injured cells.

Collectively, it was concluded that LRCs are renal progenitor-like tubular cells that provide regenerating cells, which actively proliferate and eventually differentiate into epithelial cells during tubular regeneration. Thus, intrinsic adult renal stem cells might also contribute to tubular repair. It may be possible to regenerate renal

tubules in vivo through their activation or direct delivery to sites of disease.³⁹

Fibroblasts

Renal fibrosis is a common denominator of most chronic renal diseases that progress to end-stage renal failure and is associated with major alterations in the tubular interstitial com- partment. Increased numbers of fibroblasts producing excessive amounts of extracellular matrix molecules characterize interstitial pathology. Whether interstitial fibrosis is a cause or a consequence of renal pathology remains unclear. Recently, it was hypothesized that expansion of the cellular content of the interstitium could be an attempt to restore an embryonic environment supporting the repair of injured tubules.⁴⁰Understanding the origin of interstitial fibroblasts may well contribute to our understanding of the role of these cells in renal pathology.

One hypothesis for the origin of interstitial fibroblasts proposes an epithelial–mesenchymal transition in the local formation of fibroblasts from organ epithelium. These transitions are par- ticularly apparent during fibrogenesis: Fibrotic tissue repair after injury. Strutz et al.⁴¹ first demonstrated de novo expression of a murine fibroblast-specific marker (FSP-1) in selected tubular epithelial cells during late stages of renal fibrogenesis. Ng et al.⁴²showed that progressive renal failure is associated with the transdifferenti- ation of tubular cells into myofibroblasts. A second hypothesis for the origin of adult fibroblasts argues that marrow stromal cells are fibroblast progenitors that shuttle from the bone marrow through the circulation to populate peripheral organs. Accordingly, when circulating blood cells were labeled, they could be traced to sites of active wound healing and displaying a fibroblas- tic morphology. Bucala et al.⁴³ described a distinct population of human leukocytes that was able to differentiate into fibroblasts. In an animal wound healing model, the ability of fibroblast progenitor cells to home at sites of tissue injury and participate in scar formation was demonstrated. In a human study, Grimm et al.⁴⁴assessed the relative participation of extrarenal derived–actin positive cells in the kidney. In renal transplanted patients, kidney biopsies were taken approximately 2 months after transplantation.

The level of host-derived–actin positive cells in

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the kidney varied between 77% and 30%, indicating the existence of a circulating mesenchymal progenitor cell.

Finally, Iwano et al.⁴⁵ assessed the relative contribution of fibroblasts from both origins to the interstitial fibroblast content in normal and fibrotic murine kidneys. In the normal kidney, bone marrow-derived cells were responsible for 12% of total renal fibroblasts, whereas the remaining 88% were resident fibroblasts. In renal fibrosis, as a consequence of unilateral ureteral obstruction, these numbers were 15%

and 49%, respectively. The remaining 36% was derived from epithelial–mesenchymal transdif- ferentiation. Furthermore, they showed that these transdifferentiated cells were actively involved in the fibrosis, producing abnormal amounts and types of collagen.

Pluripotent and Committed ES Cells for Renal Repair

Can a kidney be grown from pluripotent ES cells? Theoretically, human ES cells, which are

derived from blastocysts, can form derivatives of all three germ layers and give rise to all cell types of the body.¹¹ Nevertheless, their in vivo use is limited; transplantation of human ES cells directly into adoptive hosts results in teratoma growth,⁴⁶ and they have to be initially pro- grammed and differentiated in vitro into a specific cell lineage before transplantation,^47–49 a process that does not confer purity of a single cell type.⁵⁰It remains to be determined whether human ES cells can be used as a starting material for creating complex functional three-dimensional organs in addition to their potential in generating individual cells.

Organ-specific stem/precursor cells (brain, lung, skin, kidney, etc.), which are thought to be able to directly generate many or the entire differentiated cell types in an organ, similar to hematopoietic stem cells which reconstitute the blood, may be ideal for tissue replacement (Figure 15.1). Although most adult organs, including the kidney, are mainly composed of terminally differentiated cells and the existence of an intrinsic stem cell population that sup- ports regeneration is of debate, it might be useful to track down multipotent organ stem/

MESENCHYMAL CELLS

LIGANDS(?) GFs

ECM GF-RECEPTORS

PROTO-ONCOGENES INTEGRINS

INDUCTIVE EPITHELIAL CONVERSION OF MESENCHYME

INDUCTIVE URETERIC BUD

BRANCHING MORPHOGENESIS

URETERIC BUD EPITHELIA

Figure 15.1. A simplified scheme of an embryonic renal progenitor unit consisting of metanephric mesenchymal stem cells and ureteric buds, which cross-talk via growth factors (GFs) and their receptors, molecules of the extracellular matrix (ECM) and specific integrins, proto-oncogenes and specific ligands and give rise to the differentiated cell types of the adult kidney (see text).

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progenitor cells during embryonic life in areas where organogenesis is taking place. Such fetal organ committed progenitor cells are largely restricted to reconstitution of a specific organ, but they are pluripotent in their capacity to develop into a variety of different mature cell types in the growing organ.

Mammalian Kidney Development

In early embryonic life, there is a distinct stage that represents the direct developmental origin of the mature kidney^51,52 (Figure 15.1). This stage begins at 5 weeks of human gestation when a branch of the Wolffian duct, the ureteric bud, invades the metanephrogenic mesenchyme. Mutually inductive events cause the ureteric bud to branch serially (a process termed branching morphogenesis) to form the collecting ducts, renal pelvis, ureter, and bladder trigone, whereas the renal mesenchyme undergoes mesenchymal-to-epithelial conversion to form glomeruli, proximal and distal tubules, and loops of Henlé. Only at 9 weeks of human gestation will primitive glomeruli appear. In the developing human kidney, mesenchymal cells are induced into the nephrogenic pathway to form nephrons until 34 weeks of gestation.

The existence of renal ES cells in the metanephric mesenchyme is supported by several types of evidence. Previously, the presence of a single metanephric mesenchymal cell that can generate all the epithelial elements of the nephron, excluding the collecting tubule, was established using a lineage marker.⁵³This indi- cated the presence of renal epithelial stem cells.

Moreover, recent in vitro⁵⁴and in vivo^55–57data suggest that cells residing in the metanephric mesenchyme have, under various experimental conditions, the potential to differentiate, in addition to renal epithelia, into other profes- sional cell types that participate in kidney organogenesis (myofibroblasts, smooth muscle, endothelium), as well as nonrenal derivatives including cartilage, bone, and blood.

Thus, the metanephric mesenchyme contains multipotent progenitors or embryonic renal stem cells with the ability to generate, in concert with the ureteric bud, many cell types in the mature kidney. Although the ultimate goal is the identification of a single nephrogenic stem cell that can build the entire organ, these precursor

tissues offer excellent starting material to regenerate renal structures.

Over the past few years, we have studied the transplantability of human embryonic renal precursors in murine hosts.^56,58–60Because of the difficulties in obtaining sufficient numbers of human embryos, as well as the ethical problems involved with the use of human embryonic tissue, we and others have used alternative embryonic donor tissue obtained from rodents^61–65and pigs.^56,66

Defining a Gestational Window

for Optimal Growth and Differentiation of Embryonic Kidney Progenitors Free of Teratoma Risk

If embryonic kidney precursors were to be used for transplantation, extensive differentiation into nephrons along with the organization of a collecting system required for drainage of urine must take place in vivo. That is, most of the metanephric mesenchymal cells should convert into nephron epithelia and also form glomeruli after grafting, whereas ureteric buds should undergo branching morphogenesis.

Theoretically, this could be more difficult to achieve for undifferentiated precursors derived from early embryos, which contain primarily metanephric blastema and a few ureteric buds, compared with later-gestation kidneys, which have already differentiated to a certain extent.

Additional considerations should take into account properties that are inherent to undifferentiated and predifferentiated progenitor cells, such as the ability to differentiate to several lineages⁶⁷and to undergo malignant transformation in the form of embryonic tumors after transplantation.⁶⁸

Evidence for the tremendous capacity of kidney precursors to grow and differentiate after transplantation comes from studies with murine^61–65and human tissues.^56,58–60We initially transplanted human fetal kidney fragments derived from mid-gestation pregnancies (14–22 weeks) into immunodeficient mice and showed that they exhibit rapid growth and development.^58,59 Taking into account that branching and nephrogenesis continue to occur until 34 weeks of gestation, in the outer rim of the human kidney, the nephrogenic cortex, these grafts con- tinued to mature in vivo. We then compared the

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differentiative capacity of kidney precursors obtained at different timepoints of gestation.⁵⁶ Whereas whole-organ grafting can be applied for the early kidney precursors, the development of areas of graft necrosis prevents the use of this methodology for later-gestation kidneys, and they have to be implanted in fragments, as previously described.⁵⁸ For organogenesis, the early undifferentiated kidney precursors (7–8 weeks’

human gestation) were advantageous over later- gestation kidneys, significantly growing more in size and differentiating into a larger number of mature glomeruli and tubules⁵⁶[Figure 15.2 (see color section)]. These findings are in accordance with several investigators,^61–65 which transplanted embryonic day 15 (E14–E15) mouse and rat kidney precursors and showed the latter to form mature glomeruli and tubules, as well as organized cortex and medulla.

To increase the donor pool, we have also analyzed the differentiative capacity of pig kidney precursors, obtained at different timepoints of gestation, in immunodeficient mice.⁵⁶Similar to the human transplants, we could show that grafting of early pig kidney precursors, obtained from E27–E28 embryos, achieves organogenesis and leads to better growth and differentiation compared with later-gestation pig kidneys.

Because a pig gestation affords availability of very young embryos, we could perform grafting of very early pig kidney precursors (E21–E25) in immunodeficient mice. Here, grafts did not differentiate exclusively along the nephric lineage and other differentiated derivatives such as cartilage, bone, blood vessels, and myofibroblasts, could be found. As stated before, this finding complements recent in vitro⁵⁴and in vivo⁵⁵data to suggest that cells residing in the metanephric mesenchyme are pluripotent and can transdifferentiate into nonrenal derivatives especially of mesodermal origin.

Thus, we could define a window of opportu- nity for transplantation in which successful organogenesis is achieved when applying human or porcine stage-specific kidney precursors.

To gain insight into the molecular signals that stimulate the human kidney precursors to grow and develop after transplantation, we determined to what extent their transcriptional program resembles that involved in the induction of the normal human kidney or the transformation into an embryonic kidney malignancy (Wilms’

tumor).⁶⁰ Grafts originating from a 10-week human gestation kidney were harvested at

specific timepoints and cDNA was hybridized onto nylon arrays representing 1200 genes. Gene expression profiles were compared with those obtained for developing human gestation kidneys and Wilms’ tumor. Strikingly, many of the details of the molecular program required to generate and build a human nephron after implantation into the mouse recipient were similar, in a global sense, to normal kidney induction. First, most of the “nephrogenesis” genes, classified under cell cycle regulators, transcrip- tion and growth factors, signaling, transport, adhesion, and extracellular matrix molecules, which were induced in the normal process, were also observed in the developing grafts. Aberrant gene expression in the developing transplants included a small group of molecules which function in oxidative stress and indicate possible early ischemia after the grafting procedure.

Second, comparison of temporal expression profiles demonstrated that the time course for development of normal human kidneys is similar to that found in the development of transplanted kidney precursors. Our approach could also identify specific genes, including growth factors, the expression levels of which were lower in the transplants compared with the normal kidneys at specific timepoints. Development of strategies aimed at increasing the levels of such genes might further enhance the differentiative capacity of the developing grafts.

Host Versus Donor Vascularization of Embryonic Kidney Progenitors in Host Animals

Transplanted kidney precursors rely on the development of a vascular network in situ for their engraftment and growth. To improve the ability of the grafts to sustain angiogenesis in a foreign microenvironment, it is of great importance to determine the extent of vascularization and whether it is derived from host or donor endothelial cells. Moreover, hyperacute rejection is thought to be mediated via the interac- tion of preformed antibodies circulating in susceptible hosts with antigens present on the endothelium of a xenotransplanted donor organ.⁶⁹In addition, donor endothelial cells act as antigen-presenting cells to mediate cellular rejection⁷⁰and therefore represent an immuno- logical barrier for xeno- and allotransplantation.

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Classical studies with murine metanephric tissue grown in organ culture or on the avian chorioallantoic membrane have suggested that kidney endothelia arise from angiogenic ingrowth of extrinsic vessels.⁷¹ However, when Hyink et al.⁷²grafted E11–E12 kidneys from normal mice into anterior eye chambers of host transgenic mice expressing β-galactosidase in every cell, they found β-galactosidase activity in the peripheral vessels, but not in glomerular endothelial cells. Furthermore, Robert et al.⁷³ grafted E12 kidneys immunolabeled for the vascular endothelial growth factor receptor, flk-1, into kidney cortices of the adult and newborn β- galactosidase transgenic mice. In this model, cells expressing flk-1 but lacking galactosidase activity are indicative of donor vasculogenic angioblasts. Thus, when implanted into adult recipients, the grafts exhibited only few glomeruli containing host-derived endothelium, whereas a majority of glomeruli grafted into newborns contained host-derived cells. These results suggested that rather than ingrowth of vessels into the developing kidney, endothelial precursors residing in the metanephrogenic mesenchyme give rise, at least in part, to glomerular capillaries and microcirculation of the developed kidney.^52,72 In contrast, Rogers and Hammerman⁶⁵ using a non-cross-reactive anti-mouse PECAM-1 antibody to stain for host- type endothelial cells, showed that upon xenoimplantation of E15 rat kidney precursors into recipient mice, host-derived cells could be detected in external vessels as well as in some glomeruli.

We undertook the same approach to analyze for mouse-derived endothelial cells expressing PECAM-1 in developing transplants of human and pig kidney precursors.⁵⁶Similar to Rogers et al.,⁶⁵we found positive PECAM-1 immunore- activity in external vessels as well as developing glomeruli and small capillaries of grafts of early human (7–8 weeks) and porcine (E27–E28) kidney precursors, whereas grafts of later-gestation kidneys had significantly reduced host-derived vessel counts composed mainly of external vessels. Our results suggested differential staining which was dependent on the gestational age of the donor at the time of grafting. Because later- gestation kidneys have already developed a vascular network including vascularized glomeruli (donor-derived), whereas the early kidney precursors are less vascularized, before transplantation, recipient mice have a larger contribution

to vasculogenesis of the latter including the formation of the microcirculation, after grafting [Figure 15.3 (see color section)].

It seems prudent to conclude that transplanted early human and pig kidney precursors develop as chimeric organs in which blood vessels are of both donor and host origin, whereas external vessels required for graft maintenance are mostly host-derived. The exact relationship between donor and host-derived endothelial cells in the formation of the microcirculation is currently unknown.

Functionality of Embryonic Kidney Progenitors After Transplantation

In vivo differentiation of human kidney precursors into functional mature nephrons after transplantation is critical if such were to be applicable as donor tissue in clinical practice.

Studies in mice, using fluorescein isothio- cyanate-labeled dextran as a marker of filtration into the proximal tubules, demonstrated glomerular filtration in donor nephrons of murine metanephric tissue implanted into the renal cortex of neonatal mice.⁶¹ Similarly, intra- venous injections of antilaminin immunoglobulin G into rats transplanted with fetal rat kidney tissue, resulted in labeling of glomerular basement membranes in the subcapsular grafted kidneys, confirming perfusion of the grafts.⁶²Because previous transplantation studies of renal precursors obtained from murine embryos⁶¹were not able to demonstrate that donor nephrons become incor- porated into the collecting system of hosts, Rogers et al.⁶³transplanted E15 rat kidney precursors in the omentum of rat hosts (intraabdominal grafting), so as to render possible surgical anastomosis between the ureter (a ureteric bud derivative) which develops as part of the transplant and the host’s ureter (ureteroureterostomy). After anastomosis, developing grafts were shown to produce urine and clear inulin infused into the host’s circulation. Furthermore, inulin clearance, which was shown to be increased by constant infusion of insulin-like growth factor-1 (IGF-1) into hosts, began 4 weeks’ postimplantation of E15 rat kidney precursors.⁷⁴If the time course for development of normal rodent kidneys is applicable to the development of such grafts, the enhancement of inulin clearance resulting from IGF-1 administra- tion occurred after nephrogenesis was complete (3 weeks after birth).

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We analyzed tubular function in developing human kidney grafts established in immunodeficient mice, with 99technetium-DMSA renogra- phy, the uptake of which occurs through the peritubular side.⁶⁰We could demonstrate positive uptake of the radioisotope only in tubules that matured in developed grafts. To further determine kidney functionality, we measured levels of urea nitrogen and creatinine in cyst fluid collected from cysts arising from transplants of the early human and pig kidney precursors.⁵⁶ Large cysts were mostly found in transplants established in the abdomen and therefore were not limited by the renal subcapsular space. Fluid was derived by insertion of a microcatheter into the developing renal grafts.

Average levels of urea nitrogen and creatinine were higher in cyst fluid compared with those found in the sera of transplanted mice, indicating that the human and pig transplants had produced urine. Levels of urea nitrogen and creatinine in the cyst fluid were significantly lower compared with native bladder urine. The dilute urine in the cyst fluid is compatible with a reduced capacity of the fetal kidney to con- centrate urine. Thus, similar to previous reports,^61–64we were not able to demonstrate a connection between donor human and pig nephrons to the collecting system of hosts.

However, our transplants did integrate into the host’s microenvironment and use its blood vessels, and urine was produced separately from the native kidneys. Further experimentation needs to be developed to produce adequate urinary anastomosis and diversion of blood supply to the kidney grafts sufficient to correct biochemical aberrations in a uremic individual.

Increasing the number of transplants and/or administering specific human growth factors might support functional replacement.

In Vitro Propagation of Embryonic Kidney Progenitors

Recent advances in the understanding of the molecular biology of rodent renal development have enabled separate culture of the components of the developing rat kidney, namely, the ureteric bud and the metanephric mesenchyme.

Functionally recombining subcultures of each of these embryonic precursor tissues might lead to the formation of “neokidneys.” In this con- text, Steer et al.⁷⁵ took advantage of recently

identified factors that direct ureteric bud branching morphogenesis^76,77and metanephric mesenchyme induction^78,79and showed that the isolated rat ureteric bud and mesenchyme can be recombined in vitro and the resultant structure is morphologically and architecturally indistinguishable from a “normal” embryonic rat kidney precursor. In addition, the whole rat kidney rudiment in organ culture or the cultured isolated rat ureteric bud can be parti- tioned into smaller fragments and these subfractions can be propagated through several generations. These generations do not appear different from their progenitors. The subsequent generations of isolated rat ureteric bud can be recombined with fresh rat mesenchyme.

Within the recombined rat neokidney, the ureteric buds branch into the mesenchyme and appear to induce the mesenchyme to epithelial- ize and form nephrons in a normal manner. The nascent tubular nephrons in the recombination experiments form contiguous connections with limbs of the branched UB, thereby leading to an intact aqueduct between tubule and collecting system.

Considering the limited availability of human fetal tissue, this method for subculturing and propagating whole rat metanephric rudiments in vitro might be applicable to humans and provide a large number of human kidney precursors derived from a single donor.

Clearly, porcine early kidney precursors could afford an unlimited source for renal transplantation. Furthermore, the development of a large population of pig renal primordia derived from a single progenitor could potentially be manipulated in vitro before recombination, for instance, in ways that would enhance accept- ance of the xenotransplants in immunocompe- tent hosts.

Generation of Histocompatible Kidney Progenitors Using Nuclear Transfer

Nuclear transfer denotes the introduction of a nucleus from an adult donor cell into an enucle- ated oocyte to generate a cloned embryo.¹²When transferred to the uterus of a female recipient, this embryo has the potential to grow into an infant that is a clone of the adult donor cell – a process termed “reproductive cloning.” However, when explanted in culture, this embryo can give rise to ES cells that have the potential to become

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any or almost any type of cell present in the adult body (“therapeutic cloning”). ES cells derived from nuclear transfer are genetically identical to the donor’s cells, thus eliminating the risk of immune rejection and the requirement for immunosuppression.¹²Consequently, the ability to turn cloned primordial stem cells into more complex functional structures such as kidneys would potentially overcome both immune rejection and organ shortage.

Lanza et al.⁸⁰ investigated the use of nuclear transfer to generate functional renal structures.

Renal cells obtained from an early-stage cloned bovine fetus (embryonic day 56, cloned from adult bovine fibroblasts) were used to generate functional immune-compatible renal tissues.

The cloned renal cells were expanded in vitro, seeded onto collagen-coated cylindrical poly- carbonate membranes to form renal devices, and implanted back into the nuclear donor animal without immune destruction. The embryonic precursor cells organized themselves into glomeruli- and tubule-like structures with the ability to excrete toxic metabolic waste prod- ucts through a urine-like fluid, establishing once more their high capacity to regenerate functional renal structures. Because the cloned cells were derived from early-stage fetuses, this approach is not an example of therapeutic cloning and would not be undertaken in humans. Furthermore, studies showing that cloned animals have common abnormalities, regardless of the type of donor cell or the species used, and that these abnormalities cor- relate with both subtle and gross errors in the nuclear DNA leading to aberrant gene expression, might limit this strategy for therapeutic applications.^12,81

Conclusions

Regenerative medicine is focused on the development of cells, tissues, and organs for the purpose of restoring function through transplantation (Table 15.1).²The use of stem cells as starting material offers new and powerful strategies for future kidney tissue development and engineering. Currently, adult and ES cells are investigated to replace individual cells in a diseased organ,¹⁰i.e., insulin-producing pancreatic cells for type 1 diabetes mellitus or dopamine- producing brain cells for Parkinson’s disease,

but are not close to support creation of whole organs or even significant parts of such organs.

The kidney is no exception; stem cell therapy of both renal or extrarenal origin to replace renal proximal tubular cells or glomerular mesangial and endothelial cells might delay the events leading to the deterioration of the organ and consequently decrease patients’ demands for organ transplantation. Once chronic renal failure ensues, “neokidneys” will be needed to combat organ shortage. Vascularized organ xenotransplants, which are now genetically engineered not to elicit hyperacute rejection, represent one potential option.^82,83

Organogenesis is an additional alternative.

Organogenesis of complex tissues, such as the kidney, requires a coordinated sequential transformation process, with individual stages involv- ing time-dependent expression of cell–cell, cell–matrix, and cell–signal interactions in three dimensions. Precursor tissues are composed of functionally diverse stem/progenitor cell types that are organized in spatially complex arrange- ments. When obtained at gestational-specific timepoints, the theme of temporal-spatial pat- terning of progenitor cell interactions is pro- grammed in precursor tissues leading to their optimal growth and development. The findings summarized here raise hope that translation of organogenesis for purposes of organ replacement is within reach. Nevertheless, for renal replacement therapy (“neokidney”), further experimentation needs to be developed to enhance the function of the early human and porcine kidney grafts once matured. Producing a prolonged adequate urinary anastomosis of the growing kidney and deriving of blood supply sufficient to correct biochemical aberrations in a uremic individual is an important goal. Increasing the number of transplants and/or administering specific human growth factors might support functional replacement.

Considering the limited availability of human fetal tissue, a method for subculturing and propagating whole metanephric rudiments in vitro recently developed in rats,⁷⁵ might provide a large number of human kidney progenitors derived from a single donor. Alternatively, pig early kidney precursors could afford an unlimited source for renal transplantation, provided that risk for porcine endogenous retrovirus could be eliminated.⁸⁴Large-animal models are needed to test the relevance of these strategies for transplantation in humans.

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Table 1.Published strategies for renal cell replacement using biologic material Starting materialMethodBiologic mechanismTargeted cell possibleIndicationReference Mouse adult HSCsIntravenous transplantationTransdifferentiation?Proximal tubular cellsAcute tubular necrosis36 Cell fusion? Epithelial stem cell?

Mouse adult BMDCsIntravenous transplantationBone marrow hemangioblast?Glomerular endothelial cellsAcute/subacute glomerular injury23, 26 Mouse adult MSCsIntravenous transplantationPlasticity?Glomerular mesangial cellsAcute/subacute glomerular injury32, 33 IntrarenalRenal tubular cellsAcute tubular necrosis37 Embryonic kidney Subcapsular/abdominal graftingMesenchymal-epithelial conversionNeokidneysESRD56, 58–64 precursors Branching morphogenesis (rat, pig, human)Angiogenesis Rat embryonic In vitro propagationMesenchymal-epithelial conversionNeokidneysESRD75 kidney precursors Branching morphogenesis (UB-MM[SVM1]) Bovine embryonic Nuclear transferCell organizationNeokidneysESRD80 renal cells

Artificial membrane seeding Subcutaneous transplantation

Pig proximal Renal assist deviceBioartificial kidneysAcute renal failure3, 4 tubular cellsHemofiltrationShock, sepsis Extracorporeal circuit HSCs, hematopoietic stem cells; MSCs, mesenchymal stem cells; BMDCs, bone marrow-derived cells.