Brenner and Rector's The Kidney, 8th ed.

CHAPTER 69. Stem Cells in Renal Biology and Medicine

Israel Zelikovic   Karl L. Skorecki   Daniella Magen

  

 

Overview of Stem Cells, 2215

  

 

Defining Characteristics of Stem Cells, 2215

  

 

Stem Cells and Kidney Organogenesis, 2219

  

 

Overview, 2219

  

 

Kidney Organogenesis from Embryonic Metanephric Tissue, 2219

  

 

Kidney Organogenesis from Human Embryonic Stem Cells, 2220

  

 

Kidney Organogenesis from Bone Marrow-Derived Adult Mesenchymal Stem Cells, 2220

  

 

Stem Cells in the Repair of Renal Injury, 2220

  

 

Overview, 2220

  

 

Bone Marrow-Derived Stem Cells in Renal Regeneration, 2221

  

 

Adult Intrarenal Stem Cells in Renal Regeneration, 2223

  

 

Embryonic Stem Cells in Renal Regeneration, 2224

  

 

Societal and Legal Considerations in Stem Cell Research and Therapy, 2224

OVERVIEW OF STEM CELLS

Defining Characteristics of Stem Cells

Stem cells were originally defined by the capacity for asymmetric cell division.[1] This definition refers to the process wherein the stem cell yields one daughter cell that is a replica of itself, including the capacity for asymmetric cell division, whereas the other daughter cell loses this capacity and acquires other characteristics important for specialized cellular function. After asymmetric cell division, the non-stem derivatives may generate a pool of organ system restricted transit amplifying cells with an enhanced proliferative capacity or may proceed down the progressive pathway of differentiation characterized by epigenetic and gene expression modifications that eventuate in the terminally differentiated state. More recent terminology has broadened the use of the term stem cells to cover a wider array of cell types, which contribute to organ development or have the capacity to repopulate tissues and organ systems.[2] With this broader conceptual framework, it is now possible to consider stem cells according to four different sets of defining characteristics ( Table 69-1 ).

TABLE 69-1   -- Stem Cell Defining Characteristics

Characteristic

Category

Examples

Asymmetric cell division

Yes

Adult hematopoietic stem cell

No

Embryonic stem cell

 

Cancer stem cell

Differentiation potential

Totipotent

Fertilized ovum (zygote)

Pluripotent

Embryonic stem cell

 

Embryonic germ cell

Multipotent

Adult hematopoietic stem cell

 

Multipotent adult progenitor cell

Unipotent

Epidermal transit amplifying cell

 

Intestinal crypt transit amplifying cell

Replicative capacity

Indefinite

Embryonic stem cell

 

Cancer stem cell

Finite

Adult hematopoietic stem cell

 

Embryonic germ cell

Developmental stage

Prenatal

Embryonic stem cell

 

Embryonic germ cells

 

Fetal metanephric cells

 

Fetal mesencephalic cells

Postnatal

Umbilical blood stem cells

 

Adult hematopoietic stem cells

 

Adult bone marrow mesenchymal stem cells

 

 

 

Asymmetric Cell Division

Although this first characteristic was considered a sine qua non for stem cells based on their original description in the adult hematopoietic system, not all cell types currently named as stem cells necessarily display this property (see later).

Differentiation Potential

Totipotency refers to the capacity to differentiate into all cell types of the body including the extra-embryonic tissues, placenta, and umbilical cord, a property confined to the fertilized egg itself and the cells derived from the first few cell divisions following fertilization. Pluripotency refers to the capacity to differentiate into all of the specialized cell types derived from the three germ layers (ectoderm, mesoderm, endoderm) of the developing embryo, and is a hallmark feature of embryonic stem and germ cells. Multipotency refers to the capacity to differentiate into a limited number of different cell types, often restricted to a given tissue or organ system, as in the case of adult hematopoietic or epidermal stem cells. In the case of adult hematopoietic stem cells of bone marrow origin (see later), the range of differentiation has been shown to be broader than for any other adult stem cell type—but does not reach pluripotency.[3] Unipotency refers to the capacity of a pool of progenitor cells found in many organs (see later) to repopulate a rapidly turning over population of only one cell type. It is difficult, however, to be certain whether such progenitors can be distinguished from the overall population of fully differentiated cells in a tissue with high cellular turnover.[4] Cancer stem cells in general have no differentiation potential—but in some restricted cases, anti-cancer therapy induces differentiation.[5]

Replicative Capacity

The third axis describes the capacity of the cell for indefinite or extended rounds of cell division without exhaustion by replicative senescence. As will be described later, this is a distinguishing feature of human embryonic stem cells, and is attributed at least in part to high levels of telomerase activity, which protects the integrity of chromosome ends during multiple rounds of cell division. [6] [7] This is also a feature of most cancer cell types. In contrast, human embryonic germ cells, adult hematopoietic stem cells, and most other stem cell types have extended but not indefinite replicative capacity. Extended replicative capacity enables adult stem cells to contribute to tissue and organ system maintenance throughout the lifetime of an individual, whereas terminally differentiated somatic cells display limited replicative capacity, by virtue of loss of telomerase activity ( Fig. 69-1 ). Senescence reflects the eventual loss of adult stem cell replicative capacity with advanced age, or in an accelerated fashion in a number of hereditary or acquired pathophysiologic conditions. [8] [9]

FIGURE 69-1  Stem cells are characterized by indefinite or extended replicative capacity by virtue of telomerase activity, which protects telomere integrity from attrition during repeated rounds of cell division. Loss of telomerase activity during differentiation of stem cells into somatic cells is associated with progressive loss of replicative capacity during repeated rounds of cell division, a process referred to as replicative senescence.  (Modified with permission from Stem Cells: Scientific Progress and Future Directions, NIH Report, p. C3, http://stemcells.nih.gov/info/scireport. Terese Winslow, medical illustrator.)



Developmental Versus Postnatal Origin

Stem cells can be derived either from the developing human at various stages prior to birth (developmental stem cells) or from the adult (adult stem cells), with important biological, methodological, as well as societal and ethical distinctions, which will be considered in greater detail later. Developmental stem cells contribute to embryonic or fetal development, whereas adult stem cells contribute to tissue or organ system maintenance or the response to injury.

Developmental Stem Cells

Developmental stem cells refer to stem cells derived during various stages of prenatal human development. The fertilized egg or zygote, and the subsequent group of cells that follows the first three rounds of cell division are the only stage in human development in which each constituent cell remains totipotent, and capable of reconstituting an entire organism, including both the embryonic and extra-embryonic (trophectoderm) components. The blastocyst is evident at day five following fertilization, and consists of 200 to 250 cells, of which 30 to 34 consist of the inner cell mass or epiblast and the remaining cells are the outer cell mass. It is the entire blastocyst that implanted in the uterus, following which embryonic and extra embryonic development begins. At the blastocyst stage, each cell in the inner cell mass maintains an equal differentiation potential. This pluripotent state means that each of these cells has the capacity to differentiate into derivatives of all three germ line layers (ectoderm, mesoderm, and endoderm). In normal development, these cells do not persist beyond the blastocyst stage. However, in the process of in vitrofertilization, unused frozen blasotcysts that are not destined to be implanted in the uterus, instead of being discarded, can be used to generate human embryonic stem cells from the micro-dissected inner cell mass ( Fig. 69-2 ). Once established and propagated in culture, human embryonic stem cells display two cardinal features, which render them a particularly promising source for potential future cell therapy. These properties are the capacity for unlimited rounds of replication in cell culture in the undifferentiated state (unlimited replicative capacity without senescence); and the capacity for differentiation into the specialized cell types of all three embryonic germ layers. The former property reflects the high levels of expression of the enzyme telomerase, which maintains telomere integrity through multiple rounds of cell division, [6] [7] and the latter property reflects the pluripotency of the inner cell mass cells from which human embryonic stem cells are derived. A number of gene products have been described as markers of the pluripotent state, including the transcription factors Oct-3/4, Nanog, Sox2, Rex1, and the cell surface markers SSEA-3, SSEA-4, TRA-1–60, TRA-1–81, and TRA-2–54 among others.

FIGURE 69-2  Derivation of human embryonic stem cells from products of in vitro fertilization.  (Reproduced with permission from Yu J, Thomson JA: Embryonic stem cells. Chapter 1 , p. 1 in Regenerative Medicine 2006, NIH Report, http://stemcells.nih.gov/info/scireport. Terese Winslow, medical illustrator.)

 

 

Special cell culture protocols are needed to maintain human embryonic stem cells in the undifferentiated state, and their normal tendency is to undergo spontaneous differentiation.[10] A major goal of ongoing research, is to direct this differentiation process to enable enrichment to homogeneity of a given required replacement cell type, relevant to the cell therapy indeed in question. Although very significant progress has been made in the development of such enrichment protocols, significant technical challenges remain ( Table 69-2 ), and to date no successful clinical application has been reported. Perhaps the most significant of these challenges relates to circumventing the immunologic barrier and preventing rejection. Unlike cell therapy based on adult stem cells derived in an autologous manner from the recipient, embryonic stem cells and their derivatives generally emanate from a source not related to the potential recipient, and therefore can be considered as an allogeneic graft. Research studies have confirmed the immunogenicity of human embryonic stem cells, despite their very early developmental origin. Although systemic anti-rejection immunosuppressive therapy can be utilized in a manner similar to that used in allogeneic organ transplantation, several strategies have been investigated in order to preempt such immunologic rejection in the first place (Table 69-3 ). One of the most intriguing such strategies is to derive human embryonic stem cells by somatic nuclear transfer, wherein the nuclear genome of the recipient replaces the nucleus of the egg, to create human embryonic stem cells whose genome matches that of the recipient, as illustrated in Figure 69-3 . Although the generation of zygotes by somatic nuclear transfer in human cells has been reliably demonstrated, however, human embryonic stem cells from such a source have not been produced.[11] Furthermore, it is important to note that because the first few steps in somatic nuclear transfer and in reproductive cloning are identical, appropriate legal and regulatory barriers must be in place to prevent uterine implantation of zygotes generated by somatic nuclear transfer (see later section on Societal and Ethical Considerations).


TABLE 69-2   -- Challenges in Reaching Clinical Applications of Stem Cell Therapy

Enrichment and characterization

Circumventing growth arrest and scale-up

Circumventing immune attack

Biosafety and bioethical considerations

 

 

 


TABLE 69-3   -- Strategies for Preventing or Treating Alloimmune Rejection in Stem Cell Therapy

Antirejection therapy, tolerance induction, and chimerism

Adult stem cells from autologous source

Banks for human embryonic stem cells corresponding to global MHC representation

Genetic manipulation of MHC locus

Genetic engineering to introduce tolerogenic factors

Micro-encapsulation

Vaccination

Somatic nuclear transfer

Other

 

MHC, major histocompatibility complex.

 

 

 

FIGURE 69-3  Therapeutic cloning. This refers to the generation of replacement cells by the process of nuclear transfer from the potential recipient to an enucleated donor ovum cell, in order to generate patient specific human embryonic stem cells in culture.  (Reproduced with permission from Smith AG: Embryo-derived stem cells: Of mice and men. Annu Rev Cell Biol 17:435–462, 2001.)

 

 

During the further stages of prenatal human development, primitive organ and organ system structures are formed through progressive stages of differentiation. Cells derived from fetal developmental origin such as the embryonic mesonephric duct and metanephros (see later) often show enhanced proliferative capacity, as well as the capacity to differentiate into more than one type of mature or specialized cell. Thus, cells derived from various stages of fetal development have been used as sources of cell therapy in regenerative medicine, and in this sense can also be considered as stem or progenitor cells—but with more restricted replicative capacity and differentiation potential compared to human embryonic stem cells.[12] An exception is human embryonic germ cells.[13] These cells are derived from the primordial cells of the gonadal ridge of the developing fetus, and normally are destined to mature into the adult gametes. Under appropriate culture conditions, these cells also display pluripotent differentiation, although they appear to have a limited number of population doublings in contrast to the infinite replicate capacity of human embryonic stem cells.

Adult Stem Cells

Following birth, many or most tissues in organ systems are thought to contain a sub-population of cells with the capacity for long-term self-renewal, combined with the capacity for differentiation into more mature cell types with specialized functions. Typically, adult stem cells generate an intermediate state, called the progenitor cell state, before reaching full terminal differentiation. Based on research in the hematopoietic system, adult stem cells are thought to comprise fewer than 0.01% of the total number of cells in a tissue, are dispersed throughout the tissue and sometimes more widely in the body, and respond to cues in their local microenvironment. The tremendous success of hematopoietic stem cell transplantation in the management of bone marrow failure or in conjunction with myeloablative therapy in malignancy has motivated research scientists to seek corresponding adult stem cells in other organs and in other organ systems. A powerful incentive to identify and isolate adult stem cells as a potential source of cell therapy in regenerative medicine emanates from the fact that such cells would obviate societal concerns relating to the use of cells of developmental origin (see later), and might also circumvent problems of immune rejection, if they could be obtained from an autologus patient source. The adult tissues and organ systems reported to contain stem cells includes bone marrow and peripheral blood, central nervous system, blood vessel endothelium, dental pulp, and epithelia of the skin, digestive system, cornea, retina, and liver. Controversy exists regarding the existence of true adult stem cells in the pancreas, heart, and kidney.[4] Whether adult stem cells represent remnants of developmental stem cells that persist into the adult for purposes of organ maintenance and repair, or rather represent a distinct cell type dedicated for this latter purpose is not clear.

Given their low percentage and dispersed tissue distribution, it is not surprising that isolation of adult stem cells for therapeutic usage has been technically challenging. Thus, despite some potential promising results in animal models, and circumstantial evidence based on a number of clinical observations, the existence of a source of adult stem cells sufficient for cell therapy in the context of regenerative medicine remains to be proven. Furthermore, the contribution of local resident or recruited adult stem cells to endogenous organ system maintenance and repair remains controversial in many organ systems outside of the hematopoietic system. Some investigators propose that hematopoietic stem cells or else mesenchymal stem cells of bone marrow origin may maintain the capacity in the adult to transdifferentiate into a variety of different cell types including cardiac, connective tissue, neuronal, as well as renal cells (see later) that might one day prove useful in clinical regenerative medicine. [14] [15] However, incontrovertible evidence for such a therapeutic paradigm remains elusive.

In most cases, adult stem cells are administered together with their surrounding tissue environment, without isolation as a pure stem cell population, as is the case in bone marrow transplantation. It is likely that adult stem cells make a substantial contribution to restoration of organ system function following certain forms of solid organ transplantation as well. In fact, hematopoietic stem cell and solid-organ transplantation are the only documented forms of well-proven cell therapy involving adult stem cells. Although classically considered as committed to a narrow spectrum of differentiation within the organ system, some studies have suggested the possibility that certain adult stem cells may display unexpected degrees of plasticity. Such plasticity for example, would allow bone marrow derived hematopoietic or mesenchymal stem cells, to trans-differentiate into neuronal, cardiac, or renal derivatives (see later). Mesenchymal and other multipotential adult progenitor cells have been shown in experimental animal models to differentiate into a wide variety of cell types representing all three germ layers, but do not possess the capacity to develop into every cell type as do embryonic stem cells.[15]

STEM CELLS AND KIDNEY ORGANOGENESIS

Overview

The scarcity of suitable kidneys is the main limiting factor for human renal allotransplantation and the primary reason for long-term dialysis therapy in patients with ESRD. Induction of kidney differentiation and organogenesis from autologous or heterologous tissue stem cells can potentially lead to the generation of a functioning new kidney and may ultimately serve as a therapeutic strategy in kidney transplantation.

The kidney is an anatomically complex organ with a sophisticated three-dimensional organization, which is composed of multiple different cell types whose appropriate function depends on an exquisite architectural structure permissive to coordinated cell-to-cell communication and interaction. Reconstruction of either functional nephron units or a whole kidney from the cellular level first necessitates identification of appropriate stem cells as well as the means to manipulate them to evolve into the various terminally differentiated cell types comprising the mature kidney. In addition, a unique approach is necessary to enable stem cells to evolve into the spatially and temporally appropriate renal structure.

During the past decade, two main approaches for the application of kidney organogenesis from stem cells have emerged. The first involves transplantation of committed renal progenitor cells contained within early embryonic metanephric tissue into neonates or mature hosts. The second approach employs transplantation of uncommitted stem cells—either adult bone marrow-derived mesenchymal stem cells, or embryonic stem cells—into the developing embryonic metanephros, thus providing these pluripotent stem cells with the appropriate milieu for the initial steps of differentiation and commitment toward nephrogenesis.

Kidney Organogenesis from Embryonic Metanephric Tissue (see Chapter 1 )

During mammalian embryogenesis, three pairs of renal organs develop from the intermediate mesoderm: the pronephros, mesonephros and metanephros. Only the metanephros eventually develops into the mature kidney, whereas the two other early renal organs largely degenerate. Metanephric maturation is induced by invasion of a branch of the Wolffian (mesonephric) duct, termed the ureteric bud, into the metanephric mesenchyme. Morphogenesis proceeds by reciprocal induction between the metanephric mesenchyme and the ureteric bud. Factors secreted by the ureteric bud induce mesenchymal aggregation and conversion to epithelia, whereas factors secreted by the metanephric mesenchyme induce urethral bud growth and branching. The metanephric mesenchyme, initially composed of morphologically indistinguishable mesenchymal cells, differentiates into all epithelial cells of the mature nephron, excluding the collecting duct. The distal end of the ureteric bud differentiates into the collecting ducts whereas its proximal end evolves into the ureter and renal pelvis.[16] Together, the metanephric mesenchyme and the ureteric bud eventually develop into the 26 terminally differentiated cell types comprising the mature kidney.[17]

Although a single nephrogenic stem cell has not been characterized yet, accumulating data suggests that renal stem cells are contained within the metanephric mesenchyme. Mammalian tissue fragments derived from developing renal precursors within the embryonic metanephros mesenchyme were shown to regenerate renal structures.[18] Herzlinger and colleagues first isolated cells from mouse metanephric mesenchyme that were able to generate in vitro all the different types of nephron epithelia (excluding the collecting duct), indicating that these cells were renal epithelial progenitors.[19] Subsequently, metanephric mesenchyme cells were also shown to differentiate in vitro into cells other than renal epithelia, including myofibroblasts, smooth muscle and endothelium, suggesting that the metanephric mesenchyme contains pluripotent embryonic renal stem cells.[20]

In view of the fact that the metanephric tissue contains renal stem cells, transplantation of fetal metanephros can be viewed as a therapeutic stem cell application. Metanephros transplantation, originally developed as an experimental model for research in the fields of renal embryology and transplantation immunology, [21] [22] was later employed in experiments aimed at developing means to augment host renal function. The first experiments of metanephros transplantation for renal host augmentation were performed by implanting embryonic metanephric tissue into the renal cortex of neonatal mice less than 24 hours old. Metanephric grafts differentiated into functional, urine producing, nephrons containing vascularized glomeruli, mature tubuli and collecting ducts, thus demonstrating the feasibility of adding functioning nephrons to mammalian kidneys in species in which there is ongoing nephrogenesis postnatally.[23] Further progress was achieved by metanephros transplantation from embryonic day 15 rats into the omentum of non-immunosupressed, partially nephrectomized adult rat hosts. Omental grafting resulted in enhanced transplant growth to a diameter approximately one third of the native kidneys, vascularization by arteries originating from the omentum and formation of mature glomeruli, tubules, collecting systems, and ureters. The transplanted metanephroi developed into miniature kidneys showing very low, although measurable, inulin clearance.[24] Incubation of metanephroi with a cocktail of growth factors such as insulin-like growth factors, transforming growth factor-α, hepatocyte growth factors, vascular endothelial growth factor and others prior to implantation increased inulin clearance by more than 100-fold as compared to non-growth factor incubated transplants.[25] In a similar fashion, metanephroi from embryonic day 28 pigs implanted into the omentum of unilaterally nephrectomized adult pigs grew and differentiated without the need for host immunosuppression.[26]

One of the most important advantages of allogeneic metanephros transplantation over conventional adult kidney transplantation is the diminished immunogenicity of the pre-vascularized transplant during a specific time window, rendering host immunosuppression therapy unnecessary.[27] The mechanisms accounting for the reduced immunogenicity of prevasculaized embryonic metanephric tissue during early stages of development include absence of antigen presenting cells,[28] reduced expression of major histocompatibility class I and II antigens,[29] and reduced expression levels of cytokines, chemokines, and cytolytic effector molecules as compared to adult grafts.[30] In addition, most of the vascular support of transplanted metanephroi originates in the host, resulting in chimeric organs that are largely protected from host immune responses directed against transplant endothelial antigens.[31]

The combination of human allogeneic donor kidney shortage (both from adult and most probably from embryonic and fetal origin in the future) with the attenuated immunogenicity of metanephric tissue renders the option of embryonic kidney xenotransplantation an appealing potential alternative. Xenogenic metanephros transplantation has been successfully performed from human or pig into SCID mice, without the need for host immunosuppression.[12] In immunocompetent hosts, xenogenic metanephros transplantation from concordant species (rat to mouse)[31] as well as across highly disparate barriers (pig to rodent)[26] is feasible, although still mandates host immunosuppression therapy to prevent transplant rejection.[27] Successful metanephros xenotransplantation from pig to primate has not been demonstrated as yet.

For metanephros transplantation to become feasible in the foreseeable future, several requirements must be met: the metanephric transplant must provide sufficient function to sustain life in the long term, and the risks of transmitting infectious agents through xenotransplants must be eliminated, appropriate immunosuppression regimens should be developed, and the ethical and political issues coupled with use of human embryonic metanephric tissue must be addressed.

Kidney Organogenesis from Human Embryonic Stem Cells

Blastocyst-derived embryonic stem cells provide a potentially unlimited source for highly differentiated cells and tissues. Under appropriate culture conditions, ES cells can differentiate into embryoid bodies containing derivatives of all three germ cell layers[32] (see earlier). Exploring pathways for embryonic stem cell-derived renal differentiation may help to pinpoint the ideal time for harvesting a specific renal precursor cell type and may lead the way to discovering appropriate renal stem cells, which can be used for neo-organogenesis. The exact culture conditions necessary to further drive differentiation toward kidney have not yet been established[33]; however, some preliminary progress has been made. Thus, human embryonic stem cells show the ability to differentiate into kidney structures, in vivo, when grafted into immunocompromised mice to form teratomas. [6] [34] [35] [36] Similarly, mouse embryonic stem cells cultivated as embryoid bodies can spontaneously differentiate in vitro into tubule epithelial and podocyte-like renal cell types expressing renal marker molecules such as nephrin, podocin, wt-1, and Tamm-Horsfall protein.[37] Recent data indicates that embryonic stem cells can respond to metanephric inductive signals manipulating them to integrate into the developing kidney and differentiate into mature renal epithelia. Steenhard and colleagues[38]microinjected mouse ES cells that were tagged with β-galactosidase as a lineage marker into embryonic day 12 to 13 metanephric culture that mimics the microenvironment of the developing kidney. After 3 to 5 days in organ culture, embryonic stem cell derivatives were observed in epithelial structures resembling tubules. Many of these structures also expressed proximal tubule markers and Na+, K+-ATPase indicating a highly differentiated epithelium. Conversely, embryonic stem cells were observed rarely in the structures resembling glomeluar tufts. Using a combination of several nephrogenic factors including retinoic acid, Activin-A, and Bmp7, Kim and Dressler[39] induced cultured embryonic stem cells to express specific markers of intermediate mesoderm, the embryonic kidney precursor, and to form epithelial structures in vitro. When injected into mouse embryonic kidney organ cultures, treated ES cells gave rise to tubular epithelial cells with very high efficiency.

Kidney Organogenesis from Bone Marrow-Derived Adult Mesenchymal Stem Cells

Adult bone marrow-derived stem cells may serve as a safer alternative to embryonic stem in terms of therapeutic applications because they carry a lower risk for putative teratogenesis. In addition, autologous bone marrow stem cells may reduce the risk of unfavorable immune responses and can avoid ethical and political controversies.[40]

Recently, Yokoo and colleagues[41] successfully demonstrated the use of adult bone marrow derived human mesenchymal stem cells (hMSCs) in the generation of metanephroi within rodent whole-embryo culture. Based on the finding that hMSCs derived from adult bone marrow maintain plasticity and can differentiate into various cell types, depending on their microenvironment,[42] these cells were injected into in vitro cultured rodent whole-embryos at the site of renal organogenesis, allowing for the initial step of commitment toward nephrogenesis. To distinguish the donor-derived cells from the host cells, hMSCs were labeled with the LacZ gene. After 48 hours of whole embryo culture, metanephroi were dissected from embryos and subjected to further organ culture. This culture combination enabled the generation of chimeric kidneys composed of both rodent derived cells as well as of LacZ-positive hMSCs. hMSCs developed into morphologically identical cells to endogenous renal cells and were shown to express podocyte and tubular epithelial cell specific genes.[41] Further progress was recently made by the same group after transplanting the developing chimeric kidney, derived from hMSC and rodent early metanephric tissue, into the omentum of uninephrectomized adult host rats. This modification enabled the transplants to enlarge and to develop into neo-kidneys with host-derived vascular supply. hMSC-derived LacZ-positive cells were identified throughout the neo-generated kidneys and were morphologically identical to resident renal cells. Finally urea nitrogen and creatinine rich fluid was collected from expanded ureters, suggesting that the neo-kidney may produce urine. These findings suggest that hMSC can differentiate into a mature renal structure with the potential to replace lost kidney function.[43]

STEM CELLS IN THE REPAIR OF RENAL INJURY (see Chapter 29 )

Overview

Acute kidney injury (AKI) due to ischemic or chemical injury occurs in up to 5% of hospitalized patients[44] and is a major cause of morbidity and mortality. [45] [46] Allograft AKI, which occurs in up to 50% of donor kidney transplants, significantly reduces short-term and long-term graft survival.[47] Despite major efforts in investigating the pathogenesis of AKI and intensive searching for new therapies for this condition, very little progress has been made in improving its outcome. It is clear that the capacity of the kidney to regenerate functional tissue following an episode of acute injury is a major determinant of outcome of patients with AKI. There has been no specific therapy that can improve the rate or effectiveness of the repair process following acute renal injury.

An ischemic or toxic renal injury results in cell necrosis, apoptosis, and detachment of cells, which lead to denudation of the tubular basement membrane.[48] The repair process is accomplished by new cells migrating into the region and reconstituting a functional tubular epithelium. [48] [49] This tissue regeneration may result from growth factor-dependent dedifferentiation of surviving tubular cells followed by their migration, proliferation, and redifferentiation into normal tubules; migration of stem/progenitor cells residing in the interstitium of the kidney into the region of injury and their differentiation into tubular epithelial cells; or recruitment of cells that originate from outside the injured kidney (e.g., bone marrow cells) that facilitate the renal repair either by secreting factors that enhance resident renal stem cell proliferation or by “transdifferentiation” into tubular cells. [48] [49] [50] [51]

The better understanding of the mechanisms involved in kidney repair has led to the conclusion that introducing supplementary cells into a damaged kidney might aid in the repair and regeneration of the injured tissue by accelerating and augmenting the ongoing natural healing process. [49] [52] Indeed, experimental data has been interpreted to indicate that adult stem cells, either derived from the bone marrow or of renal origin, may participate in cellular repair and tissue remodeling following renal injury and might therefore serve as a tool to manage AKI. [49] [52] [53] [54] [55] [56] Also, several recent studies have demonstrated the potential role of human embryonic stem cells in renal tissue repair. [38] [39] [56]

In the following section we will summarize the accumulating evidence for the potential role of bone marrow-derived stem cells, intrinsic renal stem cells, and embryonic stem cells, in the structural and functional recovery following acute renal injury.

Bone Marrow-Derived Stem Cells in Renal Regeneration

The two major categories of bone marrow-derived stem cells (BMSCs) include hematopoietic stem cells (HSCs) and marrow stromal cells (also known as mesenchymal stem cells; MSCs). The HSCs include the precursors of all the cellular blood elements (including polymorph nuclear leucocytes, T cells, B cells, macrophages, megakaryocytes, and erythrocytes). The MSCs, whose exact function has not been well defined, are thought to play a role in promoting the survival and maturation of HSCs.

Numerous studies have shown that both HSCs and MSCs have the plasticity to cross “lineage boundaries” and differentiate into various types of cells in the body. There is evidence that HSCs differentiate into non-blood cells such as endothelial cells, hepatocytes, or pancreatic islet cells,[49] although it is not clear whether this potential reflects fusion or the HSCs with an existing cell or the true capacity of transdifferentiation. MSCs can differentiate into mesenchymal cells such as chondrocytes, osteocytes, and adipocytes.[57] Moreover it has been suggested that BMSCs contribute to regeneration of organs such as brain, liver, and heart.[52]

Trans-Differentiation of Bone Marrow-Derived Stem Cells into Renal Cells

Several studies have examined the capacity of BMSCs to transdifferentiate into renal cells. Imasawa and colleagues[58] transplanted bone marrow from mice transgenic for green florescence protein (GFP) into lethally irradiated B6 mice. The authors demonstrated that GFP-positive bone marrow cells localized to glomeruli of recipient B6 mice and acquired morphological and functional characteristics of glomeruler mesangial cells. Similarly, Masuya and colleagues[59] showed that a purified single hematopoietic (Lin-, Sca-1+, c-kit+, CD34-) cell clone was capable of differentiating into mesangial cells in irradiated recipient mice. Furthermore, the authors demonstrated by Y-chromosome testing that the process did not involve cell fusion. Two other studies, however, investigating the plasticity of HSCs by rigorously testing the in vivo cell fate of purified HSCs transplanted into irradiated animals[60] or by using the model of parabiotic mice,[61] failed to show the contribution of HSCs to renal tissue. Both studies led to the conclusion that transdifferentiated HSC progeny were not involved in kidney maintenance. The discrepancies between the findings of all these studies may be related to different populations of donor bone marrow-derived cells, differing detection methods of donor cells, different models used, or other factors.

As discussed earlier, Yokoo and colleagues[41] injected cultured human MSCs into kidney of intact human mouse embryo during whole embryo culture. They found that the MSCs incorporated into the developing tubules and glomeruli, and expressed tubule- and glomerulus-specific markers, respectively. The ability of the MSCs to differentiate into complex kidney structure was further augmented when the MSCs were induced to express the embryonic kidney growth factor glial-derived neurotrophic factor (GDNF), which is normally required for epithelial-mesenchymal signaling in the metanephric mesenchyme.

Contribution of Bone Marrow-Derived Stem Cells to Renal Repair

Several recent studies have explored the capacity of BMSCs to contribute to renal parenchymal maintenance and repair. Poulsom and co-workers[62] and Gupta and associates[63] examined kidney biopsies from male patients who had received kidney transplants from female donors. They found within the tubules Y-chromosome positive cells that co-expressed epithelial markers (CAM5.2, cytokeratin) indicating that the likely source of these tubular cells was the recipient's bone marrow. These cells were not simply infiltrating white blood cells because they did not express markers of nucleated hematopoietic cells (CD45).[63] Furthermore, in the study by Gupta and colleagues,[63] Y-chromosome positive cells were detected only in kidneys from patients with acute tubular necrosis following transplantation suggesting that the tubular injury attracted BMSCs to the kidney during regeneration.

These clinical studies were supported by several animal studies demonstrating bone marrow-derived cells in the kidney. Poulsom and associates[62] examined kidneys of female mice that had received a male bone marrow transplantation following irradiation. Approximately 8% of tubular epithelial cells contained a Y-chromosome and a smaller percentage of Y-chromosome containing cells were observed within glomeruli with morphology and location appropriate for podocytes. Ito and colleagues[64] transplanted bone marrow of transgenic rats carrying enhanced green florescence protein (EGFP) into wild-type rats. Recruitment of BMSCs into the recipients' glomeruli was significantly facilitated in response to mesangiolysis evoked in anti-Thy1 antibody-mediated glomerulonephritis. Rookmaaker and colleagues[65] used a rat allogenic bone marrow transplant model to demonstrate that BMSCs participate in glomerular endothelial and mesangial cell turnover and contribute to microvascular repair in anti-Thy1 glomerulonephritis.

Two groups have studied the contribution of bone marrow stem cells to renal regeneration in mouse models of kidney ischemia-reperfusion (I/R) injury. [66] [67] Lin and co-workers[66] transplanted a purified preparation of lineage-uncommitted HSCs (Lin-, Sca-1+, c-kit+) from male Rosa26 mice that express β-galactosidase constitutively, into lethally irradiated female non-transgenic mice after unilateral renal I/R injury. Four weeks after transplantation, donor-derived cells, detected with X-gal staining and antibody co-staining, were found primarily in the S3 segments of proximal tubules, where most injury and regeneration occurred. The finding was supported by PCR detection of the male-specific Sry gene and fluorescence in situ hybridization (FISH) detection of the Y-chromosome in the kidneys of female recipients. Using a similar model of sorted HSCs from Rosa26 mice transplanted into irradiated recipient mice, Kale and colleagues[67] showed that these cells were mobilized into the circulation of the recipient mice by transient renal ischemia and home specifically to injured region of the renal tubule. In both studies, there was no evidence of bone marrow cell engraftment in control animals undergoing bone marrow transplantation without I/R injury suggesting that an inflammatory environment is required for the integration of these cells into the kidney. [66] [67]

As opposed to these studies, demonstrating the contribution of BMSCs to kidney repair following I/R injury, other studies have failed to provide evidence that BMSCs participate in such repair. Szczypka and colleagues,[68] using the model of male GFP-transgenic bone marrow transplantation into female wild-type recipient mice, showed that BMSCs incorporated into glomeruli as specialized glomerular mesangial cells. However, no donor-derived renal tubular cells were detected in uninjured kidney or kidney after folic acid-induced injury in bone marrow recipient mice. Similarly, Duffield and co-workers[69] using the model of ischemic renal injury in male to female bone marrow transplant recipient, failed to show the presence of BMSCs in the renal tubules of injured mouse kidney (see later).

Effects of Bone Marrow-Derived Stem Cells on Function of the Injured Kidney

In addition to the studies investigating the histologic and phenotypic characteristics of bone marrow-derived cells in the kidney, several groups have examined the functional effects of these cells in renoprotection and renal repair. Using the mouse model of I/R injury described earlier, Kale and colleagues[67] showed that loss of stem cells following bone marrow ablation resulted in a greater rise in BUN after renal ischemia compared with control mice, whereas infusion of lineage-negative bone marrow cells (a mix of HSCs and MSCs) after bone marrow ablation reversed this effect. Morigi and associates[70] injected MSCs of male bone marrow into female mice that had renal injury induced by cisplatin. The finding of Y-chromosome positive cells displaying tubular cell markers in the injured tubules indicated that the MSCs engrafted the damaged kidney. Moreover, injection of these MSCs markedly diminished the initial rise in BUN, minimized tubular damage, and accelerated proliferation of tubular cells following cisplatin administration.[70] Injection of HSCs failed to exert such a beneficial effect.

In another study conducted by Tögel and colleagues,[71] rats with I/R-induced AKI were infused with MSCs into the carotid artery. This therapy resulted in marked improvement in renal morphology and renal function that, however, was not associated with differentiation of the MSCs into a tubular or endothelial cell phenotype. Instead, an altered expression pattern of inflammatory mediators including a decrease in proinflammatory cytokines and an increase in anti-inflammatory factors (such as basic fibroblast growth factor and transforming growth factor-α) was noted. The authors concluded that the beneficial effect of MSCs was primarily mediated via complex paracrine actions and not by their differentiation into target cells.

Effect of Endogenous Bone Marrow-Derived Cells on Kidney Repair

Several groups have examined the effect of mobilization of endogenous BMSCs on recovery of AKI. Stokman and colleagues[72] demonstrated that mobilization of HSCs from the bone marrow with stem cell factor (CSF) combined with granulocyte colony-stimulating factor (G-CSF) resulted in significant enhancement of functional recovery of the I/R kidney. Nevertheless, this effect was not due to increased incorporation of bone marrow-derived cells into the injured tubule. Similar to Tögel and co-workers,[71] these authors concluded that altered inflammatory kinetics (and not stem cell transdifferentiation) was responsible for the observed protective effect. Iwasaki and colleagues[73]demonstrated that mobilization of bone marrow cells into the renal tubule by G-CSF combined with macrophage colony stimulating factor (M-CSF) accelerated the improvement in renal function and prevented the renal tubular injury induced by cisplatin. In contrast to these studies, Tögel and co-workers[74] showed that mobilization of bone marrow cells with G-CSF and cyclophosphamide increased the severity of ARF in mice subjected to I/R injury presumably by enhanced kidney inflammation secondary to G-CSF-induced granulocytosis.

It is thus apparent that the true contribution of BMSCs to renal parenchymal maintenance and repair remains controversial and enigmatic. There is conflicting evidence concerning the specific subset of BMSCs (HSCs versus MSCs) participating in this process, the in vivo capacity of these cells to integrate into kidney parenchyma, their ability to transdifferentiate into tubular epithelial cells, and finally, the exact mechanism whereby BMSCs exert their effect on kidney regeneration. For example, some studies indicate that even if adult BMSCs have the capacity to differentiate into (or fuse with) renal tubular cells, it is an uncommon event and may not have a predominant role in tubular regeneration. [49] [52]

The accumulating data supports several possible roles for BMSCs in kidney repair after acute injury ( Fig. 69-4 ).[49] First, BMSCs can transdifferentiate into small numbers of tubular epithelial cells or vascular endothelial cells. Second, BMSCs can secrete factors that promote proliferation and migration of intrarenal stem cells to the injured tubule (see later). Third, BMSCs can protect tubular cells from damage/death by an endocrine effect or by suppression of inflammatory processes. Finally, BMSCs may have a direct or paracrine effect on tubular cells that protects them and promotes their proliferation. The relative roles of each of these putative mechanisms of BMSCs action on renal regeneration is a subject for future studies.

FIGURE 69-4  Possible roles for bone marrow stem cells in kidney repair after acute kidney injury. Bone marrow stem cells (BMSCs) can differentiate into small numbers of tubular epithelial cells, peritubular vascular endothelial cells, or both (1). BMSCs can secrete factors that may augment the capacity of resident renal stem cells to proliferate and enter the tubule during the repair process (2). BMSCs can act to prevent tubular cell death or enhance proliferation (or both) by an endocrine effect on the tubular cell itself, or by suppression of inflammatory responses (3). BMSCs that enter the kidney and surround the injured tubules could act in a paracrine or direct fashion to mediate cell protection and proliferation (4).  (Redrawn with permission from Cantley LG: Adult stem cells in the repair of the injured renal tubule. Nat Clin Pract Nephrol 1:22–32, 2005.)

 

 

Adult Intrarenal Stem Cells in Renal Regeneration

Considerable evidence exists for the presence of organ-specific multipotent and even pluripotent adult stem cells in several organ systems. [75] [76] [77] Adult stem cells, which are involved in organ maintenance and repair after injury, have been detected in organs characterized by rapid self renewal such as the hematopoietic system, skin, and intestinal epithelia as well as in organs with slow rates of cell turnover such as the nervous system, prostate, and liver. [75] [76] [77] Because the kidney has the capacity to regenerate and repair, as evidenced by its slow rate of cellular proliferation and functional recovery following ischemic injury, [48] [49] several investigators have explored whether adult stem cells exist in the kidney and whether these cells participate in the regeneration observed after renal ischemia.

It has been proposed that such a contribution of potential adult intrarenal progenitor cells to renal repair and regeneration may recapitulate some aspects of embryonic kidney development. As discussed earlier, during renal organogenesis, cells of the metanephric mesenchyme are induced by the ureteric bud to differentiate into specific renal lineages, thereby giving rise to the glomerular epithelium, proximal tubule, loop of Henle, and distal convoluted tubule.[78] Persistence of these cells in the adult renal tissue would provide a reservoir of tubule cell progenitors that could migrate into the tubule and differentiate into epithelial cells in response to renal injury.[49] Multipotency of such adult progenitor cells, similar to the multipotency that is characteristic for single cells in the metanephric mesenchyme,[20] could lead to regeneration of mesangial cells, endothelial cells, and other cell types in the damaged adult kidney.

Adult Stem Cells in the Normal and Injured Kidney

In searching for stem cells in the adult kidney, Oliver and colleagues[79] used the properties of these cells of self renewal and slow cycling time. Cells with these characteristics can be distinguished by retention of a nucleotide label such as bromodeoxyuridine (BrdU), which is incorporated into the DNA of cells during DNA synthesis. The investigators, who injected newborn mice with BrdU and analyzed their kidneys several months later, found numerous BrdU-labeled cells in the interstitial and tubular components of the renal papilla.[79] The number of these cells decreased after transient renal ischemia indicating that they were likely involved in renal repair. Staining for Ki67, a marker of proliferation, provided evidence for their proliferative capacity. Isolation and in vitro culture showed that these papillary cells, similar to metanephric mesenchymal stem cells,[20] showed remarkable plasticity and co-expressed epithelial and mesenchymal markers. These findings led to the conclusion that the renal papilla is a niche for adult kidney stem cells. Using a similar method, Maeshima and associates[80] demonstrated the presence of slow cycling cells in normal rat kidney. The authors showed that during tubular regeneration following renal ischemia, slow cycling cells act as a source of regenerating cells that have an immature phenotype, actively proliferate, and consequently differentiate into epithelial tubular cells.

Two recent studies examined the relative importance of intrinsic renal cells and BMSCs in kidney regeneration. [69] [81] Lin and co-workers[81] produced transgenic mice that expressed EGFP specifically in mature renal tubular epithelial cells. Following I/R injury, EGFP-positive cells showed incorporation of BrdU and expression of vimentin indicating that the cells participated in tubule regeneration and were derived from renal tubular epithelial cells. In bone marrow cell-transplanted mice that were subjected to I/R injury, the majority (89%) of the regenerating cells were descendents of either surviving tubular epithelial cells or potential renal stem cells. In contrast, bone marrow cells made only a minor contribution to tubular repair. Duffield and colleagues[69] created mice with chimeric bone marrow between wild-type and GFP or β-gal mice. After renal ischemic injury, a large number of BMSCs were observed in the interstitium but not in the renal tubule of these mice. Similarly, in female mice with male bone marrow chimerism, no Y-chromosome+ cells were detected in the tubules after ischemic injury. On the other hand, many tubular cells expressed proliferating cell nuclear antigen, which is reflective of a high proliferative rate of endogenous surviving tubular cells. The conclusion of these two studies was that intrarenal cells and not BMSCs were the main source of renal repair after ischemic injury.

Identification and Isolation of Adult Intrarenal Stem Cells

Several studies have purported to characterize and isolate the potential intrarenal stem cell population by using various specific cell surface markers. The approach of Challen and colleagues[82] was to identify specific genetic markers of the uninduced metanephric mesenchyme (MM) by cDNA micro-array analysis, comparing gene expression in the uninduced MM with that of more rostral derivatives of the intermediate mesoderm. Using this method, the investigators identified 21 genes preferentially expressed by the uninduced MM at the point of commitment to a metanephric lineage. Two of these, CD24 and cadherin 11 genes, encode surface proteins that might be useful for isolation of progenitor cells from the adult kidney. Bussolati and colleagues[83] used the human CD133+ stem cell antigen as a selection marker for potential renal progenitor cells. This molecule, expressed in hematopoietic stem cells, was shown to be also expressed by endothelial progenitor cells, by undifferentiated intestinal epithelial cells and by embryonic kidney.[84] CD133+ cells derived from normal adult human kidney (which also expressed PAX2, an embryonic renal marker) were capable of expansion and limited self renewal and differentiation in vitro into epithelial or endothelial cells.[83] On subcutaneous implantation in SCID mice, the undifferentiated cells formed tubular structures expressing renal epithelial markers. Intravenous injection of fluorescently labeled, renal-derived CD133+ cells in SCID mice with glycerol-induced rhabdomyolysis and ARF, resulted in homing of these cells into the injured kidney and integration into the tubules. The investigators concluded that these cells represented a multipotent adult resident stem cell population participating in the repair of renal injury.

Hishikawa and co-workers[85] explored a specific subset of renal stem cells termed side population (SP) cells, which they recognized by their known ability to extrude Hoechst 33342 dye and showed that these cells were located in renal interstitium. The authors demonstrated that these cells expressed musculin/MyoR, a member of basic helix-loop-helix transcription factor family. Intravenous infusion of kidney SP cells counteracted the decrease in GFR in mice with acute tubular injury induced by cisplatin administration.[85] Moreover, a musculin/MyoR-dependent rise in renoprotective growth factors such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) was evident in SP cells isolated from the injured kidney.

The exact nature of the intrarenal stem cells explored in the studies discussed earlier has not been defined. It is currently unknown whether these cells are truly undifferentiated and their complete developmental potential is unclear. An interesting study in this context is a recent morphological work by Vogetseder and colleagues,[86] who demonstrated that BrdU-retaining (and therefore slow cycling “stem”) cells in the rat renal tubule display features of fully differentiated tubular cells including the expression of the Na+, K+-ATPase and the Na+-phosphate cotransporter. The investigators' hypothesis, based on their findings, was that differentiated tubular cells, rather than multipotent adult stem cells, are self-renewing in the healthy kidney. This study is in concert with the study by Dor and colleagues,[4] who by using genetic lineage tracing, demonstrated that preexisting, fully differentiated b cells were the major source of new pancreatic b cells during adult life and after pancreatectomy in mice. These studies suggest that terminally differentiated cells, at least in organs with relatively slow turnover such as the pancreas and the kidney, retain a significant proliferative capacity in vivo. These findings open a new avenue for research on the origin of replenishment of the injured kidney.

Future studies on stem cells in the kidney in health and after injury will assist in their isolation and analysis throughout nephrogenesis and will provide further insight into the identity and nature of the putative adult renal stem cells.

Embryonic Stem Cells in Renal Regeneration

As discussed earlier, embryonic stem cells can develop into all types of cells in the body, and a number of proofs of principle have been demonstrated in the repair of tissue injury in animal models.[87] Recent studies have explored the capacity of embryonic stem cells to differentiate into renal tissue in vitro and to integrate into kidney compartments in vivo. [6] [33] [35] [38] [39] [55] Schuldiner and colleagues[33] have demonstrated the ability of several growth factors to induce human embryonic stem to differentiate in vitro into renin and WT1 expressing cells. Human embryonic stem cells have the capacity to differentiate into kidney structures when injected into immunosupressed mice to form teratomas.[6] Steenhard and co-workers injected embryoid body-derived mouse embryonic stem cells into embryonic day 12 to 13 mouse metanephric kidneys in culture, and identified embryonic stem cell-derived LacZ-positive cells in epithelial structures resembling tubules, and on rare occasions, in structures resembling glomerular tufts.[38] Using a combination of retinoic acid, activin A, and bone morphogenetic protein-7, embryoid bodies were induced to express specific markers of intermediate mesoderm and to form epithelial structures in vitro.[39]

Recently, Kobayashi and associates[35] stably transfected mouse ES cells with Wnt4 cDNA by culturing them with hepatocyte growth factor (HGF) and Activin A and induced them to differentiate into aquaporin-2 expressing embryoid bodies (EB). Transplanting the Wnt4-EBs in the mouse renal cortex resulted in aquaporin 2-expressing, tubule-like formations.

The therapeutic potential of self-renewing, pluripotent embryonic stem cells as a virtually unlimited donor source for tissue repair and cell transplantation is obvious. Nevertheless, many theoretical, practical, methodological, and ethical problems must be solved before ES cell technology can be applied in the management of renal disease.

The multi- or pluripotency of stem cells, which enables them to cross lineage boundaries, their homing into (BMSCs), presence in (intrarenal stem cells), or transformation to (ES cells) renal parenchyma, and finally, the accumulating evidence that stem cells participate in kidney maintenance and regeneration, render all types of stem cells worthy of further research as potential sources for cellular repair following acute renal injury. Nevertheless, numerous issues remain unsettled, and much additional research is needed before stem cell therapy for the damaged kidney is transferred from a concept to reality. Future studies are needed to understand the biology of stem cells in the kidney and the mechanisms controlling their mobilization, proliferation, and differentiation. The relative roles of BMSCs and intrarenal progenitor cells in kidney protection and repair, the identification of the specific subtypes of stem cells with the highest biological potency (and, therefore, therapeutic potential), and the suitable cell surface markers to be used for the isolation and purification of these cells are important topics for future investigations. Future studies should provide more insight into the exact mode of action of stem cells in renal regeneration and into the relative importance of processes such as transdifferentiation, fusion, and endocrine/paracrine effects in stem cell action. Strategies using various growth factors and pharmacologic agents should be developed to enhance the regenerative capacity of intrarenal progenitor cells. Finally, research should be conducted to define the optimal dose, frequency, and the route of application of externally administered stem cells.

Notwithstanding all the remaining questions, stem cell therapy, once well defined and fully established, may become a very powerful tool in achieving the cure of acute renal failure and, possibly, chronic renal failure as well as a variety of other kidney diseases.

SOCIETAL AND LEGAL CONSIDERATIONS IN STEM CELL RESEARCH AND THERAPY

Societal and ethical, and legal considerations have appropriately emerged as major issues in developmental stem cell research and therapy. [88] [89] [90] [91] [92] [93] This is also a major distinction between developmental and adult stem cells, based on different views of personhood status prior to birth. In particular, there is a broad worldwide consensus that full personhood sanctity and associated rights should be accorded to each individual from at least the moment of birth irrespective of sex, sexual orientation, religious beliefs, and ancestral or population affiliation, physical capacity, or age. However, this consensus does not apply to the status of an individual prior to birth, and differences of opinion are often based on cultural settings and religious beliefs. Table 69-4 provides the list of those milestones in human development from the gamete stage until birth, which are relevant to the different views of when full personhood sanctity and rights are achieved. Many societies, as well as secular and religious legal codes, recognize at least three such milestones as being critical in the assignment of the rights and protections in comparison with the postnatal state.


TABLE 69-4   -- Human Developmental Milestones Relevant to Societal and Ethical Considerations in Stem Cell Research

Haploid state: Sperm and egg

Fertilization: In vitro[*]

    In vivo[*]

Uterine implantation[*]

  

 

Embryonic and fetal development

  

 

Twinning potential[*]

  

 

Recognizable human form[*]

  

 

Heartbeat

  

 

Ossification

  

 

Fetal movement

  

 

Extra-uterine viability[*]

Birth[*]

 

*

Milestones that have been used in different societies to define points of increasing or full personhood status.

 

The first of these milestones is that of uterine implantation. According to many but not all ethical viewpoints and legal codes, the pre-implantation embryo or product of IVF is not assigned full personhood rights and protection, and this constitutes the legal basis for permitting derivation of embryonic stem cells from IVF-derived blastocysts. The rationale for this demarcation leads to the notion that the pre-implantation blastocyst represents potential life, whose actualization is dependent on parental consent and subsequent technical intervention. The milestone of uterine implantation has also emerged as particularly important in religious legal codes. Thus even those religious belief systems including Islam, Judaism, and certain Christian denominations, which restrict abortion of an in utero embryo or fetus, do sanction use of the pre-implantation blastocysts for research and therapeutic purposes. However, the Catholic church and certain other Christian denominations, accord full personhood sanctity and protection from the moment of conception, and forbid development of human embryonic stem cells using currently available approaches, which involve blastocyst destruction. Accordingly, a number of scientific and technological innovations have been proposed for derivation of human embryonic stem cells without destruction of potential viable blastocyst. [94] [95] Such innovations include utilization of “defective” blastocysts with no chance for subsequent viability and derivation of human embryonic stem cells from single cells at the morula stage following fertilization. However these innovations have not yet reached a level of full technical validation or uniform ethical acceptance. Most recently, there have been scientific reports of the induction of the pluripotent state in mouse fibroblasts by the introduction of a series of only four “pluripotency switch” genes. [96] [97] [98] [99] Extrapolation of this approach to differentiated cells of human origin, together with examination of biosafety considerations (non-tumorigenecity) is eagerly awaited. A second milestone involves the post-implantation transition from the embryo to the fetus with recognizable organ system features, at approximately 10 to 12 weeks of gestation. Certain secular and religious legal codes permit the termination of pregnancy and utilization of the subsequent products of such aborted pregnancies for developmental stem cell and research and therapy, whereas others forbid this approach. There are differences in the approach to the use of spontaneously aborted embryos or fetuses in research or for therapeutic applications, usually reflecting a broader but not uniform level of permissiveness among individuals and societies. A third critical milestone is the achievement of stage of potential extrauterine viability. This milestone has been advanced through earlier and earlier stages of gestation, now occurring as early as 22 to 24 weeks following conception. Finally, as noted earlier, the moment of birth is considered by virtually all societies, cultures, and religious systems to mark state of full personhood status along with its attendant rights and protections.

There are constituencies around the world that have adopted policies or enacted laws that reflect different opinions regarding personhood status at each of these milestones. Thus, for example in the United States, blastocysts derived from in vitro fertilization may be used in stem cell research and therapy; however, federal funding for research involving human embryonic stem cells is restricted to a specific subset of stem cell lines that were already in existence prior to August 9, 2001. In contrast, in the United Kingdom, Canada, France, Israel, and some other countries federal funding may be used to support such research. In contrast, in Germany, Italy, and certain other countries, derivation of human embryonic stem cells is prohibited under criminal law. The United Nations Educational, Scientific, and Cultural Organization (UNESCO) has also addressed this issue and provided a general set of non-binding guidelines, which are permissive to human embryonic stem cell research, at least using existing cell lines. Even those constituencies, cultures, and religious belief systems that do sanction the derivation of human embryonic stem cells from IVF blastocysts, condition such use upon the appropriate regulatory oversight and parental consent. Another important bioethical consideration relates to the notion of the “slippery slope”. Bioethicists and legislators are often concerned about the possibility that research scientists and clinicians will move beyond allowable activities into prohibited domains. A prime example relates to somatic nuclear transfer, which is a first step shared in therapeutic cloning and reproductive cloning ( Table 69-5 ). Because of the overlap in the first steps, certain constituencies have prohibited somatic nuclear transfer, based on the fear of illegitimate extrapolation to reproductive cloning. Most bioethicists feel that appropriate legislation, regulation, and enforcement can preempt inappropriate research and clinical activities that might result from a “slippery slope” effect.


TABLE 69-5   -- Differences Between Therapeutic and Reproductive Cloning

 

Therapeutic Cloning

Reproductive Cloning

End product

Cells growing in a Petri dish

Human being

Purpose

To treat a specific disease of tissue degeneration

Replace or duplicate a human

Time frame

A few weeks (growth in culture)

9 months

Surrogate mother needed

No

Yes

Sentient human created

No

Yes

Ethical implications

Similar to all embryonic cell research

Highly complex issues

Medical implications

Similar to any cell-based therapy

Safety and long-term efficacy concerns

Adapted from Vogelstein B, Alberts B, Shine K: Please don't call it cloning. Science 295:237, 2002.

 

 

 

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