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Adult Stem Cells for Tissue Repair A New Therapeutic Concept?

Martin Krbling, M.D., and Zeev Estrov, M.D.
From the Department of Blood and Marrow Transplantation (M.K.) and the Department of Bioimmunotherapy (Z.E.), the University of Texas M.D. Anderson Cancer Center, Houston. Address reprint requests to Dr. Krbling at the University of Texas M.D. Anderson Cancer Center, Department of Blood and Marrow Transplantation, Unit 423, 1515 Holcombe Blvd., Houston, TX 77030, or at mkorblin@mdanderson.org. N Engl J Med 2003;349:570-82.
Copyright 2003 Massachusetts Medical Society.

dult human stem cells that are intrinsic to various tissues have been described and characterized, some of them only recently. These cells are capable of maintaining, generating, and replacing terminally differentiated cells within their own specific tissue as a consequence of physiologic cell turnover or tissue damage due to injury.1 Hematopoietic stem cells that give rise to blood cells and move between bone marrow and peripheral blood are the best-characterized adult stem cells in humans. Recent data suggest that adult stem cells generate differentiated cells beyond their own tissue boundaries, a process termed developmental plasticity. In this review we focus on in vivo models of adult stem cells derived from bone marrow and peripheral blood and their potential therapeutic applications.

adult stem cells and their potential for developmental plasticity

Stem cells are defined as cells that have clonogenic and self-renewing capabilities and that differentiate into multiple cell lineages.2 Whereas embryonic stem cells are derived from mammalian embryos in the blastocyst stage and have the ability to generate any terminally differentiated cell in the body, adult stem cells are part of tissue-specific cells of the postnatal organism into which they are committed to differentiate. Phenotypically characterized adult stem cells are listed in Table 1.1,3 The hematopoietic system has traditionally been seen as an organized, hierarchic system with multipotent, self-renewing stem cells at the top, committed progenitor cells in the middle, and lineage-restricted precursor cells, which give rise to terminally differentiated cells, at the bottom.2 However, this classic paradigm of stem-cell differentiation restricted to its organ-specific lineage is being challenged by the suggestion that adult stem cells, including hematopoietic stem cells, retain a previously unrecognized degree of developmental plasticity that allows them to differentiate across boundaries of lineage, tissue, and germ layer (Fig. 1). The hierarchical view no longer seems correct.3,4 The molecular mechanisms of lineage switches within the hematopoietic system have been studied extensively,5 but the mechanisms that determine transitions in the fate of adult stem cells remain poorly understood. The results of recent studies of the plasticity of adult stem cells, which contradict the dogma that the differentiation and commitment of adult stem cells are restricted to their own tissue, are the subject of intense discussion. These findings demand the most stringent criteria for providing conclusive evidence. To prove that stem cells derived from bone marrow and peripheral blood, including hematopoietic stem cells, are indeed transformed into solid-organspecific cells, several conditions must be met. First, the origin of the exogenous cell integrated into solid-organ tissue must be documented by cell marking, preferably at the single-cell level. Cells also should be processed with a minimum of ex vivo manipulation (e.g., culturing), which may make them more susceptible to crossing lineages. Second, the exog-

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enous cell must be shown to have become an integral morphologic part of the newly acquired tissue. Third, and most important, the transformed cell must be shown to have acquired the function of the particular organ into which it has been integrated, both by expressing organ-specific proteins and by showing specific organ function.

Table 1. Adult Human Stem Cells and Their Primary Direction of Differentiation. Cell Type Hematopoietic stem cells Mesenchymal stem cells Tissue-Specific Location Bone marrow, peripheral blood Bone marrow, peripheral blood Cells or Tissues Produced Bone marrow and blood lymphohematopoietic cells Bone, cartilage, tendon, adipose tissue, muscle, marrow stroma, neural cells

preclinical in vivo studies

Various stem-cell preparations originating from hematopoietic tissue have been used in in vivo studies of stem-cell plasticity, including unselected cells from bone marrow, purified hematopoietic stem cells, and single hematopoietic stem cells that originate from either bone marrow or peripheral blood. Because hematopoietic tissue (from both bone marrow and peripheral blood) harbors a population of heterogeneous stem cells that includes hematopoietic stem cells, mesenchymal stem cells, multipotent adult progenitor cells, and endothelial precursor cells, some of the contradictory findings that have been reported may be explained by the use of different sources and preparations of stem cells.
adult stem cells derived from bone marrow
Neural stem cells Hepatic stem cells Pancreatic stem cells

Ependymal cells, astrocytes Neurons, astrocytes, (subventricular zone) of oligodendrocytes the central nervous system In or near the terminal bile ductules (canals of Hering) Oval cells that subsequently generate hepatocytes and ductular cells

Intraislet, nestin-positive Beta cells cells, oval cells, duct cells Skeletal muscle fibers

Skeletal-muscle Muscle fibers stem cells or satellite cells

Stem cells of the Basal layer of the epidermis, Epidermis, hair follicles skin (keratibulge zone of the hair nocytes) follicles Epithelial stem cells of the lung Tracheal basal and mucussecreting cells, bronchiolar Clara cells, alveolar type II pneumocyte Mucous and ciliated cells, type I and II pneumocytes Paneths cells, brush-border enterocytes, mucussecreting goblet cells, enteroendocrine cells of the villi

Initial in vivo studies were performed with the use of marked, unselected cells derived from bone marrow. After transplantation of the cells into animals that had undergone a conditioning treatment, there was evidence that the cells formed nonlymphohematopoietic tissue, such as muscle fibers,6 hepatocytes,7 microglia and astroglia,8 and neuronal tissue.9,10 Although the differentiation of stem cells derived from bone marrow into organ-specific, nonlymphohematopoietic cells has been described, the origin of stem cells responsible for the formation of nonlymphohematopoietic tissue remains obscure. Thus, to prove that stem cells from bone marrow and, in particular, hematopoietic stem cells are capable of forming solid-organ tissue cells, purified stem cells with a specific marker had to be transplanted and shown to generate specific functional tissue cells.11 Donor-derived muscle fibers that express dystrophin have been identified after sex-mismatched transplantation of purified and phenotypically characterized hematopoietic stem cells.12 Lagasse and colleagues13 went one step further by providing proof of functionality: in an animal model of ty-

Stem cells of Epithelial cells located the intestinal around the base of epithelium each crypt

rosinemia type I, transplantation of as few as 50 purified hematopoietic stem cells restored both the hematopoietic and biochemical liver functions in the recipient by correcting the genetic aberration. In a similar fashion, hepatocytes that express human albumin were identified in immunodeficient mice in which purified human hematopoietic stem cells had been transplanted.14 In other studies, purified stem cells, including hematopoietic stem cells, have been shown to generate functioning cardiomyocytes and vascular structures,15,16 as well as neointimal smooth-muscle cells and endothelial cells that contribute to arterial remodeling in various models of vascular lesions.17 Further in vivo evidence that hematopoietic stem cells are capable of multifunctional differentiation was provided by Krause and colleagues.18 Using a limiting-dilution technique, they showed that single cells differentiated into mature hematopoietic

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Endoderm

Epithelial cells of liver, lungs, GI tract

Blastocyst

Mesodermal progenitor cell

Myocytes, osteoblasts, chondrocytes, adipocytes, endothelial cells

Mesoderm Embryonic stem cells Hematopoietic progenitor cell

Bone marrow cells, blood cells

Ectoderm

Keratinocyte precursors, neurons, oligodendrocytes, ependymal cells

Figure 1. Model of Embryonic and Adult Stem-Cell Differentiation along and across Germ-Layer Boundaries. Embryonic stem cells differentiate into three germ-layertype cells when cultured under appropriate conditions. Some data suggest that postnatal mesodermal hematopoietic progenitor cells destined to differentiate along their own pathway may, like embryonic stem cells, cross germ-layer boundaries to generate endodermal or ectodermal tissue cells. GI denotes gastrointestinal.

cells and into mature epithelial cells of the skin, lungs, and gastrointestinal tract. However, because of the localization of the purported transdifferentiated progeny and the lack of evidence of organ-specific function of those cells, the physiologic relevance of the reported observations is not clear. In similar experimental settings, transplantation of a single hematopoietic stem cell or cultured cells derived from a single hematopoietic stem cell resulted not only in hematopoietic engraftment but also in retinal neovascularization19 and the generation of functioning glomerular mesangial cells.20
adult stem cells derived from peripheral blood

ing factor and recombinant human granulocyte colony-stimulating factor were shown to contribute to ocular neovascularization in mice23 and neovascularization of ischemic myocardium in rats,24 respectively. Orlic and coworkers25 provided evidence of the generation of cardiomyocytes in a murine model of myocardial infarction after a cytokine-induced increase in the concentration of circulating stem cells. These changes resulted in improved ventricular function and survival.25 However, in a different experimental setting involving a nonhuman primate model of myocardial infarction and reperfusion, cytokine-induced mobilization of stem cells did not result in a favorable outcome.26

Since progenitor cells derived from bone marrow can reach the target solid organ through the peripheral blood,21,22 determining whether peripheralblood stem cells follow a differentiation pathway specific to solid organs, similar to that seen for stem cells derived from bone marrow, was the logical next step. Several investigators have reported that circulating human stem cells, mobilized into the peripheral blood by cytokine administration, contribute to the generation of nonlymphohematopoietic tissue. Endothelial progenitor cells mobilized by recombinant human granulocytemacrophage colony-stimulat-

clinical in vivo studies

adult stem cells derived from bone marrow and peripheral blood

Because the experimental conditions in a preclinical setting are currently far more sophisticated than those available in a clinical setting, the mechanisms underlying the reported clinical observations should be interpreted with caution. The same is true when extrapolating knowledge about stem-cell function in nonprimate species to humans. The ability of human progenitor cells derived from bone marrow to generate nonlymphohemato-

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poietic tissue has been studied in allogeneic sexmismatched transplants. The initial clinical studies were performed by Horwitz and colleagues,27 who claimed that stem cells derived from bone marrow led to improved osteogenesis in children with osteogenesis imperfecta. Subsequently, two groups independently reported the presence of donor cells that were positive for the Y chromosome in liver tissue after male-into-female bone marrow transplantation or female-into-male liver transplantation. Theise and colleagues28 reported the presence of donorderived hepatocytes in liver tissue at levels ranging from 4 to 43 percent and cholangiocytes at levels ranging from 4 to 38 percent. At the same time, Alison et al.29 also reported donor-derived hepatocytes, although at lower levels (0.5 to 2 percent). In another study of patients who underwent liver transplantation, Kleeberger et al.30 confirmed livertissue chimerism by genotyping cells that had been microdissected with a laser and immunolabeled with cytokeratin, using DNA analysis of highly polymorphic short tandem repeats. Cholangiocyte chimerism was an almost universal finding in the early period after transplantation, whereas hepatocyte chimerism tended to occur later, coinciding with recurrent hepatitis. Epithelial cells of the esophagus, stomach, small intestine, and colon of donor origin were reported at a frequency of 0.4 to 4.6 percent in patients who underwent sex-mismatched bone marrow transplantation in whom graft-versus-host disease or ulcer formation subsequently developed.31 Translating their observations in rodents to a clinical level, Mezey et al.32 identified rare clusters of donor-derived neuronal cells containing the Y chromosome at a frequency of up to 7 per 10,000 neurons in the hippocampus and cerebral cortex of recipients of bone marrow transplants. In a similar transplantation setting, donor-derived Purkinje neurons were identified in the brains of adults who had received bone marrow transplants.33 Indications that circulating stem cells can contribute to the formation of solid-organ tissue derive from studies of solid-organ transplantation. Quaini et al.34 and Mller et al.35 have reported male chimerism in heart allografts from female donors. In addition to the effect of direct migration of cardiomyocytes from adjacent recipient tissue into the allograft, the researchers postulate that circulating stem cells from the transplant recipient contribute to ventricular remodeling in heart allografts. Similarly, Lagaaij et al.36 have documented endothelialcell chimerism in patients with rejection of renal al-

lografts. Circulating donor-derived endothelial cells have been identified in the peripheral blood of recipients of stem-cell transplants by determination of their origin either from transplanted circulating angioblasts or from transplanted stem cells that differentiated in vivo into endothelial cells.37 We and our colleagues recently reported the presence of XY-positive hepatocytes and epithelial cells in five female recipients of peripheral-blood stemcell allografts mobilized by recombinant human granulocyte colony-stimulating factor from male donors.38 Donor-derived, XY-positive, nonlymphohematopoietic cells were identified at frequencies ranging from 0 to 7 percent in the skin, gut, and liver of all five female stem-cell recipients as early as day 13 and up to day 354 after transplantation with allogeneic stem cells. Although two conditions for proof that donor-derived male peripheral-blood cells contributed to the formation of solid-organ tissue in female recipients were met with the use of the Y-chromosome marker and morphologic evidence of donor cells integrated into the recipients solidorgan tissue, we did not demonstrate that donorderived cells, whether located within mature or immature cell compartments, expressed functions specific to solid-organ tissue. Donor-derived keratinocytes that were positive for the Y chromosome and cytokeratin were found at a frequency ranging from 3.7 to 14.8 percent in six patients who underwent transplantation of sexmismatched peripheral-blood stem cells.39 However, when the same epidermal skin cells were cultured over a period of 18 to 32 days through multiple passages to eliminate any contaminating lymphohematopoietic cells, Y-chromosomespecific sequences from the donor-derived cells could not be detected. One might speculate that the donorderived cells found in skin tissue were artifacts of the engulfment of the recipients skin cells by donor macrophages; that cells bearing the Y-chromosomespecific sequences were lost after several passages in culture; or that the donor cells had little or no proliferative capacity in vitro.39 Also, donorderived buccal epithelial cells were identified in recipients of sex-mismatched peripheral-blood stemcell transplants up to 4.5 years after transplantation at a frequency between 0.8 percent and 12.7 percent.40 Given the number and variety of clinical studies that have already been performed (Table 2), and the lack of data from studies of large animals in particular, primates caution should be exercised in predicting a potential clinical benefit.

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Table 2. Potential Clinical Applications of Hematopoietic TissueDerived Adult Stem Cells for Tissue Repair or Replacement.* Route of Cell Application Intravenous

Disease or Category Osteogenesis imperfecta Tyrosinemia type I (liver) Hepatitis B or C

Model Clinical BMT (allogeneic)

Stem-Cell Source Bone marrow cells

Outcome

Study

Increased total-body bone mineral Horwitz et al.27 content Correction of metabolic liver disease Profoundly reduced viremia in vivo Lagasse et al.13

Experimental BMT Purified bone marrow Intravenous derived hematopoietic stem cells Experimental BMT Interferon-btransfected or peripheralbone marrowderived blood SCT or peripheral-blood derived stem cells Experimental BMT HGF-transfected bone or peripheralmarrowderived or blood SCT peripheral-blood derived stem cells Intravenous

Eto and Takahashi41

Liver cirrhosis

Intravenous

Inhibition of fibrinogenesis and apoptosis, resolution of hepatic fibrosis Reduction of infarcted area, improved cardiac hemodynamics Generation of donor-derived cardiomyocytes and endothelial cells Decrease in infarct size and mortality, increase in ejection fraction, improvement in hemodynamics

Ueki et al.42

Myocardial infarction

Experimental BMT Purified bone marrow Intracardial derived hematopoietic stem cells Experimental BMT Purified bone marrow Intravenous derived hematopoietic stem cells G-CSFinduced experimental stem-cell mobilization Experimental G-CSF mobilized peripheralblood SCT Clinical BMT (autologous) Clinical BMT (autologous) Clinical BMT (autologous) Peripheral-blood stem cells NA

Orlic et al.15

Jackson et al.16

Orlic et al.25

Mobilized and purified human peripheralbloodderived angioblasts Bone marrow cells

Intravenous

Stimulation of neovascularization Kocher et al.24 and angiogenesis in infarcted region

Intracoronary

Decreased infarct size, improved ventricular function and myocardial perfusion

Strauer et al.43

Purified bone marrow Intramyocardial Enhanced left ventricular function, Stamm et al.44 derived hematopoietic improved infarct tissue perstem cells fusion Bone marrow or peripheral-blood cells Bone marrow cells Bone marrow cells Intracoronary infusion Improved left ventricular ejection fraction, improved regional wall motion in infarct zone Assmus et al.45

Ischemic heart disease

Clinical BMT (autologous) Clinical BMT (autologous)

Intramyocardial Improved myocardial perfusion and function Intramyocardial Improved myocardial perfusion and left ventricular function Intravenous Improvement of aging-impaired cardiac angiogenic function

Tse et al.46 Perin et al.47 Edelberg et al.48

Experimental BMT Bone marrow cells Impaired cardiac angiogenic function associated with aging Chronic limb ischemia Ischemic vascular disease Clinical BMT (autologous) GM-CSFinduced experimental stem-cell mobilization Bone marrow cells

Intramuscular Improvement in anklebrachial injection in index, pain at rest, and painischemic leg free walking time Improved neovascularization of ischemic tissues

Tateishi-Yuyama et al.49 Takahashi et al.23

Peripheral-bloodderived NA endothelial progenitor cells

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Table 2. (Continued.) Route of Cell Application

Disease Category Ischemic retinopathy

Model

Stem-Cell Source

Outcome Improved retinal angiogenesis

Study Otani et al.50

Experimental BMT Purified bone marrow Intravitreal derived hematopoietic stem cells and endothelial progenitor cells Experimental BMT Single hematopoietic stem cells Intravenous

Induction of retinal neovascularization Partial restoration of dystrophin expression in the affected muscle Partial restoration of dystrophin expression in the affected muscle Generation of alveolar type II pneumocytes

Grant et al.19 Gussoni et al.12

Intravenous Duchennes muscu- Experimental BMT Purified bone marrow derived hematopoietic lar dystrophy stem cells Clinical BMT (allogeneic) Bone marrow cells Intravenous

Gussoni et al.51

Lung diseases with Experimental BMT Single hematopoietic stem cells extensive alveolar damage Renal diseases involving glomerular mesangial tissue Neurodegenerative diseases

Intravenous

Krause et al.18

Experimental BMT Clonal population of cells Intravenous derived from a single hematopoietic stem cell Experimental BMT Bone marrow cells Experimental BMT Bone marrow cells Clinical BMT (allogeneic) Clinical BMT (allogeneic) Bone marrow cells Bone marrow cells Stem cells derived from umbilical-cord blood

Generation of glomerular mesan- Masuya et al.20 gial cells

Intraperitoneal Generation of cells expressing neuronal markers Intravenous Intravenous Intravenous Intravenous Generation of cells expressing neuronal markers Generation of cells expressing neuronal markers Possible formation of Purkinje neurons Improved functional recovery from neurologic deficit

Mezey et al.9 Brazelton et al.10 Mezey et al.32 Weimann et al.33 Chen et al.52

Experimental Middle-cerebralcord-blood artery occlusion transplanta(stroke) tion

* Experimental denotes data drawn from nonhuman settings, BMT bone marrow transplantation, SCT stem-cell transplantation, G-CSF granulocyte colony-stimulating factor, GM-CSF granulocytemacrophage colony-stimulating factor, HGF hepatocyte growth factor, and NA not applicable. Patients underwent revascularization with the use of balloon angioplasty and subsequent stent implantation before infusion of bone marrow cells. Patients underwent coronary-artery bypass grafting before infusion of bone marrow cells.

limitations in identifying chimeric cells

The interpretation of the data on tissue chimerism could be hindered by technical conditions. Y-chromosomepositive nuclei must be unequivocally identified as belonging to nonlymphohematopoietic solid-organspecific cells integrated into female tissue, in order to rule out the possibility that donorderived inflammatory cells, such as infiltrating lymphocytes or macrophages, are mistakenly identified as solid-organspecific cells.53 One way to demonstrate that Y-chromosomepositive nuclei are truly

associated with solid-organspecific cells is to perform concomitant or sequential staining, including fluorescence in situ hybridization staining, on the same thin tissue section instead of staining serial sections separately.38

reports that question developmental plasticity

Two reports have been published that question the potential of cells derived from hematopoietic tissue

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to undergo cell fate transition and in so doing contribute to cell formation in solid-organ tissue. Castro et al.54 failed to detect neural-like cells in the brains of mice that received purified stem cells derived from bone marrow or unfractionated bone marrow cells. Similarly, after transplanting single hematopoietic stem cells into conditioned recipient mice, Wagers et al.55 found very few donor-derived cells in nonlymphohematopoietic tissue. As discussed in detail elsewhere with regard to the findings of Castro et al. and Wagers et al., the interpretation of negative results depends on the experimental system in which the hypothesis is tested and on its specificity and sensitivity.56-58

vironment for hybrid cells and may not be representative of other solid-organ tissues.65 Thus, cell fusion appears to account for the presence of cells in solid-organ tissue that show donor characteristics to some extent, but not completely. Fusion could even be seen as a physiologic ongoing repair mechanism by which cells deliver healthy and new genes to highly specialized cells to prevent them from dying and to correct genetically defective cells.66 Distinguishing between stem-cell fate transition and cell fusion demands that researchers use rigorous criteria in studying the phenomenon of stem-cell plasticity.

cell fusion
Recently, the validity of stem-cell plasticity as a mechanism for generating nonlymphohematopoietic tissue has been questioned. Some have suggested that previously overlooked cell fusion may explain the findings in the studies described above. Confirming earlier studies performed under rather nonphysiologic culture conditions,59,60 two in vivo studies of mice with a fatal metabolic liver disease have provided evidence that bone marrow cells from normal donors generate healthy hepatocytes by forming hybrid cells that contain both donor and host genes.61,62 Most of the fused cells had tetraploid or hexaploid DNA content. In contrast, cytogenetic analysis of bone marrowderived cells specific to solid organs in preclinical allogeneic transplant studies revealed a diploid karyotype.20,63 Donor-derived, solid-organspecific cells in patients who have received allogeneic bone marrow or peripheral-blood stem-cell transplants have also been identified as diploid,28,29,38,39,64 except in liver tissue, where polyploidy is not uncommon. Furthermore, numerous cytogenetic analyses of bone marrowbiopsy specimens from patients undergoing allogeneic stem-cell transplantation have been characterized by euploidy, except in diseased tissue. It is possible that hybrid cells undergo a reduction division, thus converting the hyperploid cell to a diploid karyotype and thereby concealing the fusion history.61 However, detection of donor-derived, diploid XY-chromosomepositive cells in recipient solid-organ tissue as early as 9 days15 and 13 days38 after allogeneic cell infusion makes this explanation unlikely. It has also been discussed whether hyperploid hepatocytes may provide a favorable en-

models of differentiation of adult stem cells into solid-organspecific cells

The results of transplantation studies involving sex-mismatched peripheral-blood stem cells suggest that the cells responsible for generating solidorganspecific cells are, like hematopoietic stem cells, a group of circulating mononuclear cells. As outlined by Frisen,67 these studies of peripheralblood stem-cell transplantation suggest four possible explanations for how adult stem cells derived from bone marrow or peripheral blood differentiate into nonlymphohematopoietic tissue cells (Fig. 2). The first explanation is that multiple distinct types of stem cells circulate, with each type differentiating into its own lineage-restricted tissue. This deterministic model is supported by the fact that various progenitor cells with clonogenic potential circulate in the peripheral blood, including hematopoietic stem cells, mesenchymal stem cells,68 endothelial precursor cells,36,37,69 skeletal stem cells,70 and smooth-muscle progenitor cells.71,72 Therefore, it is conceivable that nonlymphohematopoietic, organ-specific stem cells, like hematopoietic stem cells, move between their own solid tissue and the peripheral blood. The second explanation postulates that a primordial equivalent to the embryonic stem cell, located in the bone marrow, peripheral blood, or both and available throughout adulthood, gives rise to various circulating, lineage-restricted stem cells. Evidence of such an adult somatic stem cell has been provided recently by Jiang et al.,73 who isolated multipotent adult progenitor cells from bone marrowderived cultured cells; the progenitor cells were negative for CD34, CD44, CD45, c-kit, and major-histocom-

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Multiple lineagerestricted stem cells

Adult somatic stem cells

Transdifferentiation of lineage-restricted stem cells

Dedifferentiation of mature cells followed by redifferentiation

Figure 2. Various Models for Generating Solid-Organ Tissue Cells through Differentiation of Bone MarrowDerived and Circulating Adult Stem Cells. In the first model, distinct stem cells differentiate, each into its own organ-specific cell (Panel A). In the second model, primitive somatic stem cells located in hematopoietic tissue differentiate into various organ-specific cells (Panel B). In the third model, stem cells, such as hematopoietic stem cells, differentiate along their predetermined pathway. Under certain, probably rare conditions, tissue injury or another stimulus causes some stem cells to deviate from their predetermined pathway and generate cells of a different tissue a process known as transdifferentiation (Panel C). In the fourth model, mature cells dedifferentiate into cells with stem-celllike characteristics and eventually redifferentiate into terminally differentiated cells of their own tissue or a different tissue (Panel D).

patibility-complex (MHC) classes I and II. These progenitor cells maintain their functional capacities when cultured for more than 100 population doublings, without obvious senescence, and differentiate in vivo into the hematopoietic lineage and into epithelium of the liver, lung, and gut. However, function of these end-differentiated cells within specific solid organs has not been proved. According to the third hypothesis, a circulating hematopoietic stem cell that expresses a default pathway of hematopoietic differentiation deviates, under certain conditions, from its preprogrammed pathway of differentiation and jumps the lineage barrier (a phenomenon known as transdifferentiation). Preclinical data support this hypothesis.18-20 The fourth explanation is that a differentiated, solid-organspecific cell regains characteristics similar to those of a stem cell in order to generate, through peripheral blood, differentiated cells of another tissue specificity (dedifferentiation and redifferentiation).3,74,75 In vivo dedifferentiation of epidermal cells to islands of stem cells in response to recombinant human epidermal growth factor has been reported.76 None of these four models of stem-cell differen-

tiation have yet been proved to explain the mechanism underlying developmental stem-cell plasticity. In our opinion, preclinical data favor the transdifferentiation model, although this remains controversial. More than one model may be valid.

potential role of circulating adult stem cells in tissue repair

It is well known that circulating hematopoietic stem cells are critical for hematopoietic homeostasis.77 Similarly, one may hypothesize that circulating stem cells contribute to homeostasis in solidorgan tissue.
mechanisms of stem-cell recruitment and differentiation

The mechanisms by which circulating stem cells are recruited into various solid-organ tissues and tissuespecific cells are subsequently generated are not fully understood. Tissue injury that causes changes in the microenvironment may play an important part in stem-cell recruitment (Fig. 3). In this regard, the highest percentage of donor-derived hepatocytes

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Skin Liver Endogenous stem cells (oval cells) Injury

Injury

Hepatocytes Injury

Growth and differentiation factors Exogenous blood-derived hematopoietic stem cells

Endogenous stem cells (crypt cells)

Intestine

Figure 3. Possible Roles of Bone MarrowDerived and Circulating Stem Cells in the Repair of Solid-Organ Tissue. After tissue injury, stem cells that are intrinsic to the tissue replace necrotic cells as a first line of defense. If the pool of endogenous stem cells is exhausted, exogenous circulating stem cells are signaled to replenish the pool and participate in tissue repair. Thus, circulating stem cells may serve as a backup rescue system.

(about 40 percent) was found in a liver-transplant recipient with fibrosing cholestatic recurrent hepatitis C.28 A low frequency of hepatocyte replacement by bone marrowderived stem cells has also been reported (up to 2.2 percent of total hepatocytes), even in the absence of histologic evidence of acute hepatic tissue injury,78 suggesting that circulating cells take part in tissue homeostasis. Because the model for most experimental and clinical transdifferentiation studies is allogeneic stem-cell transplantation with use of the Y chromosome as a marker, human leukocyte antigen disparity cannot be ruled out as a trigger for circulating stem-cell transdifferentiation. A concentration of stem cells at the site of tissue damage has been shown to contribute to tissue repair. As noted earlier, Orlic and colleagues reported the repair of infarcted cardiac tissue after either the

injection of stem cells derived from bone marrow directly into heart tissue surrounding the infarct15 or a systemic increase in the number of peripheralblood hematopoietic stem cells to a level approximately 250 times as high as the base-line level with use of a combined treatment of recombinant human granulocyte colony-stimulating factor and stem-cell factor.25,79 The release of chemokines such as stromalderived factor from injured hepatic tissue and interaction with its specific receptor, CXCR4, on hematopoietic precursor cells is another prototype of how tissue repair may function by aiding in the recruitment of circulating hematopoietic precursor cells to the site of tissue injury.80 On a molecular level, transcriptional regulation of hematopoietic stem cells may be altered in response to signals from the local environment. Spe-

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cifically, homing, growth, and differentiation factors that may be involved in transitions of cell fate have been described for neuronal cells as a prototype.3
a clinical model of solid-organ tissue generated by circulating stem cells

Anderson and colleagues11 hypothesized that under physiologic steady-state conditions, an equilibrium is maintained whereby endogenous stem cells intrinsic to a specific tissue and, to a much lesser extent, blood-derived stem cells of heterologous lineage replenish apoptotic tissue. However, a rigorous demand for tissue repair that cannot be met by endogenous stem-cell differentiation may, as a backup system, trigger circulating stem cells to undergo differentiation into solid-organ tissue, though this is believed to be rare under physiologic steady-state conditions. Thus, the pool of tissue-intrinsic stem cells can be considered an intermediary between systemically circulating stem cells and terminally differentiated solid-organspecific cells (Fig. 3). Experimental evidence of a stepwise developmental progression from adult bone marrow through a stem cell intrinsic to muscle tissue to an adult muscle fiber in response to injury was provided by LaBarge and Blau.63 Adult cells derived from bone marrow contributed to the formation of up to 3.5 percent of differentiated muscle fibers. The triggering mechanisms for the plasticity of bone marrow-derived stem cells seemed to be an insufficient pool of stem cells intrinsic to this tissue in concert with an increased demand for the production of new cells and changes in the microenvironment as a result of tissue injury at the site.

cessible, as is the case with the circulating stem-cell pool that is routinely used for harvesting hematopoietic stem cells.81 Second, the concentration of stem cells at the site of tissue regeneration must be sufficient, which can be accomplished either by cytokine-induced mobilization of hematopoietic stem cells,81 mesenchymal stem cells,59 angioblasts,36,69 and smooth-muscle progenitor cells71,72 from extravascular sites into the circulating blood or by directly delivering the cells to the site of tissue injury. Third, appropriate signals from the site of damaged tissue must direct exogenous stem cells to the site where they are needed. However, we do not yet clearly understand how to manipulate the microenvironment surrounding the area of tissue regeneration actively and signal exogenous blood-derived stem cells to participate in tissue regeneration in vivo. As with transplantation of hematopoietic stem cells, solid-organ tissue repair can be accomplished by differentiation of autologous or allogeneic stem cells. Allogeneic transplants are advantageous in experimental settings in which the Y chromosome is used as a marker, as well as in hereditary stem-cell disorders, in which transplantation of normal stem cells is supposed to generate normally functioning tissue. Differentiation of autologous bone marrow or circulating stem cells into solid-organ tissue seems the preferred therapeutic approach, as it precludes immunologic disparities. It should be emphasized that the interpretation of some of the clinical data reported so far is controversial, and in some cases there is no confirmation of their validity. Selected potential clinical indications for generating solid-organ tissue with the use of stem cells derived from hematopoietic tissue are listed in Table 2.

potential clinical applications embryonic stem cells

There are essentially two strategies for using adult stem cells derived from hematopoietic tissue for tissue repair. One approach is based on identifying and expanding in vitro multipotent adult progenitor cells that, like embryonic stem cells, are capable of generating mesodermal, ectodermal, and endodermal tissue.73 The other approach is based on the in vivo availability of a pool of systemic and circulating adult stem cells that can be manipulated to generate or repair solid-organ tissue. Assuming that circulating stem cells generate cells specific to solid organs in vivo, a potential clinical concept of tissue repair would require three conditions. First, the stem-cell pool must be easily acMultipotent adult progenitor cells are similar to embryonic stem cells in that they can be extensively expanded in vivo, and have extensive proliferation and clonal multilineage differentiation potential73 in vitro and in vivo to form cells of three germ layers. In contrast to embryonic stem cells, multipotent adult progenitor cells can be harvested from autologous bone marrow, thus avoiding the rejection that results from the expression of MHC proteins in human embryonic stem cells.82 Immunologic disparities associated with the use of embryonic stem cells may be overcome by applying the technique of somatic-cell nuclear transfer (therapeutic

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cloning). A major concern with the use of embryonic stem cells for therapeutic purposes is their potential to form teratomas in vivo; such tumorigenicity is thus far unknown for pluripotent adult progenitor cells.83,84 The in vivo differentiation potential of circulating adult stem cells into nonlymphohematopoietic tissue under steady-state conditions seems to be inferior to that of ex vivo expanded embryonic stem cells and multipotent adult progenitor cells. However, the extent to which in vivo manipulation of circulating adult stem cells and their target tissue can induce terminal cell differentiation for repairing or replacing solid-organ tissue remains to be demonstrated.

conclusions and outlook

We are only beginning to understand the circulating blood, not only as a distributor of hematopoietic progenitor cells but also as a systemic supplier of progenitor cells that have the potential to participate in the homeostasis of various solid-organ tissues and that are capable of nonlymphohematopoietic tissue repair. There is a growing body of evidence that the adult stem-cell system may be more flexible
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We are indebted to Kate O Suilleabhain and David Galloway for their editorial assistance.

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