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Stem cells and diabetes
Dossier: Stem cells
Biomed Pharmacother 2001 ; 55 : 206-12 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0753332201000506/FLA
Stem cells and diabetes G. Berná1, T. León-Quinto1, R. Enseñat-Waser1, E. Montanya2, F. Martín1, B. Soria1* 1 Institute of Bioengineering, University Miguel Hernández, Campus de San Juan, E-03550, Alicante, Spain; 2Laboratory of Diabetes and Experimental Endocrinology, Endocrine Unit (13-2), CSUB-Hospital of Bellvitge, University of Barcelona, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Barcelona, Spain
(Received 22 January 2001; accepted 29 January 2001)
Summary – Diabetes mellitus is a metabolic disorder affecting 2–5% of the population. Transplantation of isolated islets of Langerhans from donor pancreata could be a cure for diabetes; however, such an approach is limited by the scarcity of the transplantation material and the long-term side effects of immunosuppressive therapy. These problems may be overcome by using a renewable source of cells, such as islet cells derived from stem cells. Stem cells are defined as clonogenic cells capable of both self-renewal and multilineage differentiation. This mean that these cells can be expanded in vivo or in vitro and differentiated to produce the desired cell type. There exist several sources of stem cells that have been demonstrated to give rise to pluripotent cell lines: 1) embryonic stem cells; 2) embryonic germ cells; 3) embryonic carcinoma cells; and 4) adult stem cells. By using in vitro differentiation and selection protocols, embryonic stem cells can be guided into specific cell lineages and selected by applying genetic selection when a marker gene is expressed. Recently, differentiation and cell selection protocols have been used to generate embryonic stem cell-derived insulin-secreting cells that normalise blood glucose when transplanted into diabetic animals. Some recent reports suggest that functional plasticity of adult stem cells may be greater than expected. The use of adult stem cells will circumvent the ethical dilemma surrounding embryonic stem cells and will allow autotransplantation. These investigations have increased the expectations that cell therapy could be one of the solutions to diabetes. © 2001 Éditions scientifiques et médicales Elsevier SAS diabetes / stem cells / transplantation
Diabetes mellitus is a heterogeneous metabolic disorder affecting 2–5% of the adult population in developed countries. Worldwide prevalence figures give an estimate of 130 million people in 2000 and 300 million in 2025. Diabetes mellitus can be classified broadly into two groups: the insulin-dependent type (IDDM or type 1), in which the treatment mainly relies on the self-injection of insulin several times daily, and the non-insulin-dependent type (NIDDM or type 2). Despite the distinct etiology et pathophysi*Correspondence and reprints. E-mail address: [email protected] (B. Soria).
ology both types may result in late complications (nephropathy, retinopathy, neuropathy, etc.). The Diabetes Control and Complications Trial [36] has shown that tight control of blood glucose can delay and diminish the progression of long-term complications; however, intensive insulin therapy (5–6 injections daily) and almost permanent blood glucose control requires highly motivated patients and does not liberate them from insulin therapy. Transplantation of insulin-producing cells isolated from donor pancreata could be a cure for type 1 and some cases of type 2 diabetes. In recent years, islet transplantation failed to materialize the hope for long-term
Stem cells and diabetes
freedom of exogenous insulin. Less than 10% of the almost 300 islet allografts transplanted since 1990 resulted in insulin independence for periods of more than 12 months [9]. A recent report by Shapiro et al. has demonstrated that using a glucocorticoid-free immnunosuppresive therapy combined with the infusion of an adequate fresh islet mass resulted in insulin independence and good metabolic control for periods of more that 12 months in seven type 1 diabetic patients [29]. However, the therapeutic potential of this approach is limited by the scarcity of transplantation material and the long-term side effects of immunosuppressive therapy. On the other hand, the potential risk of infection by animal endogenous viruses has prevented so far the therapeutic uses of islet xenografts. These problems may be overcome by deriving islet cells from stem cells. This review will concentrate on the pathway from progenitors to insulin-containing cells, either early progenitors (embryonic stem cells, ESC) or near progenitors (ductal cells). STEM CELLS: CONCEPT, PROPERTIES AND TYPES Stem cells are defined as clonogenic cells capable of both self-renewal and multilineage differentiation [42]. However, self-renewal in itself does not define stem cells and these may be non-dividing or slowlydividing cells. Some adult stem cells are commonly defined as those that can be activated to undergo proliferation upon tissue injury to achieve functional restitution. Between the stem cell and its terminally differentiated progeny there is usually an intermediate population of committed progenitors with limited proliferative capacity and restricted differentiation potential; these are the cells that display greatest proliferative activity under physiological and pathophysiological circumstances. The mechanisms of differentiation and the intermediate states can vary in different species and/or tissues, but all of them share these two properties: the capability to produce identical daughter cells (‘self-renewal’), and the possibility to produce daughters that are fated to differentiate [42]. Whilst the former means that they can be expanded, the latter indicates that they can differentiate to produce the desired cell type, either in vivo or in vitro. In terms of potentiality, they may be classified as totipotential (giving rise to any cell type including the trophoblasts of the placenta), pluripo-
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tential (when they may differentiate into any cell type including the germ line) and unipotential. Unipotential stem cells are cell lineage progenitors committed to differentiate into a particular cell type [30]. These properties offer tremendous potential for clinical application, serving as an unlimited source of cells for transplantation and tissue regeneration therapies, as well as intensive drug and toxic screening as an alternative method for animal assays. The earliest stem cell in ontogeny is totipotent (from zygote to the inner cell mass of the blastocyst); soon thereafter, totipotent cells give rise to somatic stem, progenitor cells and primitive germ-line stem cells [43]. Very little is known of the stages between somatic stem cells and the emergence of tissue- or organspecific stem cells in the neurula stage. At this stage haematopoietic stem cells appear. Since these are the best known stem cells, most of the concepts have been developed after them, though it is not clear that direct extrapolations can be made to other stem cell types. In addition, stem cells are powerful tools for studying the control of in vitro cell differentiation. A number of properties besides self-renewal and differentiation potential have been ascribed to stem cells, including the ability to undergo asymmetric cell divisions, to exist in a mitotically quiescent form and to clonally regenerate [42]. However, maintenance and/or loss of the ‘stemness’ properties depend on cell autonomous regulators (transcription factors and internal clocks) modulated by external signals (secreted factors, cell-to-cell and cell-to-matrix interactions) [42]. Differentiation of ESC (obtained from the inner cell mass of the blastocyst) occurs in vitro when the growth factor signals for self-renewal are not sufficient in the culture medium. The self-renewal of undifferentiated mouse ESC depends on an exogenous supply of the cytokine leukaemia inhibitory factor (LIF) [44], which binds to LIF receptor (LIFR) and gp130 signalling complex, thereby activating STAT3 and the receptor-associated Janus kinase (JAK) [5, 22]. Together with the activation of the LIF-signalling pathway, the POU transcription factor Oct-3/4 acts as a main regulator of pluripotency that controls lineage commitment [23]. On the other hand, the exact nature of these signals is unknown for human ESC, which expresses Oct-4 but does not rely on an exogenous supply of LIF to sustain their proliferation as undifferentiated stem cells [27, 38]. In this regard, human ESC underwent low levels of
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spontaneous differentiation during routine passaging and this process was accelerated by growing cultures at high density for 4 to 7 weeks [27, 38]. Finally, spontaneous differentiation of human ESC into cell types originating from each of the three embryonic germ layers also occurs in vivo when grafting them into immunocompromised severe combined immunodeficient (SCID) mice [27, 38]. SOURCES OF STEM CELLS Pluripotent and totipotent cell lines Three main primary sources of human tissue have been demonstrated to give rise to pluripotent cell lines: 1) ESC derived from the inner cell mass of human blastocyst stage embryos [27, 38]; 2) embryonic germ cells (EGC) derived from the gonadal region of 5- to 9-week-old aborted human foetuses [28]; and 3) embryonic carcinoma cells (ECC) derived from teratocarcinoma tissue [2] (table I). A fundamental difference between these cell lines is that whilst ESC and EGC lines are genetically normal and diploid, ECC lines are transformed and are commonly aneuploid. They also differ in their cell surface marker expression, cell culture conditions, pluripotentiality and clinical utility. Thus whereas ECC lines have been of use in cell differentiation studies of early human development [2, 24, 37], ESC and EGC may also serve as a source of desired cells, or even as enucleated recipients of nuclei from patient cells instead of enucleated oocytes [35]. This procedure will lead into ‘customised’ pluripotent stem cells, which will overcome the present difficulties related to immune compatibility and graft rejection, with the subsequent use of immunosuppressive drugs. Adult stem cell lines Certain tissues maintain the capability to regenerate themselves during an entire lifetime due to the presence of stem cells. Skin, intestinal epithelia or blood cells display a high rate of cell renewal, including both cell proliferation and death, in the normal adult, even in the absence of injury. Other tissues such as liver or muscle may regenerate when injured. Finally, it is generally thought that terminally differentiated cells have suspended their differentiation possibilities and maintain a fixed phenotype with negligible
Table I. Stem cells that may potentially be used to derive pancreatic B-cells. Totipotential stem cells Embryonic stem cells Embryonic germ cells Embryonic carcinoma cells Multipotential stem cells Intestinal epithelia cells Bone marrow stroma cells Others Committed stem cells Fetal pancreatic stem cells Pancreatic duct cells Others
division potential. For many years this has been the accepted paradigm. However, recently some of the accepted paradigms have fallen into discredit. Several studies have shown that adult stem cells can be derived from tissues thought to lack regenerative capacity. Neural [14], retina [39] and bone marrow [45] are examples of tissues that have been incorporated into the category of those that possess stem cells. Furthermore, some of these adult stem cells are not irreversibly committed to a particular fate and appear to have a much broader capacity of differentiation than was previously recognised. For example, two groups have recently described the presence of multipotential adult stem cells from brain tissue of adult mice [11, 18]. These two groups have demonstrated that cells from different origins (ependymal cells [18] or astrocytes [11]) may give rise to distinct neural cells. In addition, some studies indicate that neural stem cells can produce a variety of blood cell types [3] or that stem cells isolated from muscle are capable of forming haematopoietic cell lineages [17], and haematopoietic stem cells have been shown to give rise to a variety of tissues [25]. Moreover, Clarke et al. have recently demonstrated that adult neural stem cells can contribute to the formation of chimeric chicks and mice, indicating that adult neural stem cells have a very broad developmental capacity [10]. This violation of the accepted paradigm does not preclude the search for adequate grounds that the notion which states “differentiation requires continuous regulation” [4] is fully supported. As in the case of ESC, the signals that control the maintenance and/or loss of the somatic stem cell phenotype and fate are only partially known. In addition, very little is known about molecular markers of the stem cell phenotype
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in adult tissues. In the case of epidermal stem cells, high levels of β1 integrin (an integral membrane protein responsible for cell adhesion to the extracellular matrix) are characteristic of cells with stem cell properties and β1-mediated cell adhesion suppresses the onset of terminal differentiation [41, 42]. IN-VITRO DIFFERENTIATION AND CELLLINEAGE SELECTION STRATEGIES Embryonic stem cells can be propagated indefinitely in vitro and, under certain conditions, induced to differentiate into several lineages such as cardiomyocytes (mesoderm derivative) [19], or neural precursors (ectoderm derivative) [20]. In contrast, in vitro differentiation into endoderm-derived cells, such as pancreatic B-cells, is not spontaneously produced. The sequence, combination and amplitude of the signalling mechanisms driving a stem cell into a committed pathway are not well known and are probably governed by biochemical and biophysical factors. However, the combination of cell density, growth factors and cell polarization may result in an enrichment of certain cell populations which express markers of endoderm-derived cells such as amylase, elastase, carboxypeptidase, PDX-1, insulin, glucagons, etc.). Subsequent cell lineage selection may result in highly pure cell types. Using an ‘intelligent’ transgene (figure 1, GB-1) that couples the insulin gene with the neomycin-resistant gene has made possible the selection of insulin-producing cells based on their antibiotic resistance. This cell lineage selection strategy allows multiple variants, such as using the regulatory region (figure 1, GB-2), the promoter region of the gene encoding the transcription factor PDX-1 (figure 1, GB-3) or the pancreatic polypeptide promoter (figure 1, GB-4). Both PDX-1 and PP positive cells have been postulated as B-cell precursors [16]. Furthermore, any of these precursors can be coupled to the GFP gene and use the protein fluorescence to identify and separate the desired cell line. Finally, flanking the construct with the recombinase inducible gene (Cre) allows the removal of the chimeric DNA construct after the selection of the cell type [13]. Identification of the B-cell precursor together with a more detailed knowledge of the signals that drive later steps in B-cell differentiation could, in theory, be used for in vitro growth and differentiate precursor cells taken either from aborted foetuses or pan-
Figure 1. Structure of the transgenes used in cell-lineage selection strategies. GB-1: human insulin gene-β-geo/pGK-hygror construction; GB-2: human insulin promoter-β-geo/pGK-hygror; GB-3: PDX-1 promoter-β-geo/pGK-hygror; GB-4: PP promoter-β-geo/pGKhygror.
creatic ducts. The exact nature of the pancreatic stem cell is still uncertain; several markers have been proposed to identify stem cells, including PDX-1 [31], BCL-2 [7] and cytokeratins 5 and 14 [26]. Furthermore, is not known whether B-cell precursors in the adult are distinct from foetal B-cell precursors. Replication is considered to be the principal mechanism in increasing B-cell mass after birth [40]. Both replication of preexisting B-cells and differentiation from pancreatic duct epithelium can take place in the adult life and offer a possibility for the expansion of the B-cell mass [32]. Recently, human pancreatic duct cells have been grown and induced to differentiate in vitro [6]. The recipe includes adding keratinocyte growth factor, nicotinamide and ITS medium (insulin + transferrin + selenium) during 1–2 weeks to promote proliferation, followed by coating with a murine basement membrane preparation (Matrigelt). The use of adult donor ductal cells avoids the controversy of using foetal or embryonic stem cells by using a material which, in principle, is more committed to
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Table II. Transdifferentiation events observed in the literature. From
To
Ref.
Neural cells Muscle cells Neural cells Bone stroma cells
Blood cells Blood cells Chimeric mouse Neural cells
Neural cells Bone stroma cells Bone stroma cells
Muscle cells Muscle cells Liver cells
Bjornson, 1999 [3] Jackson, 1999 [17] Clarke, 2000 [10] Woodbury, 2000 [45] Brazelton, 2000 [8] Mezey, 2000 [21] Galli, 2000 [15] Ferrari, 1998 [12] Alison, 2000 [1]
differentiate into B-cells. Unfortunately, the expansion capability is more limited and restricts its clinical utility since the total islet mass transplanted is a determinant factor for reaching insulin independence. Based in fresh islet transplantation an islet mass of approximately 12,000 islet equivalent/kg of body weight is required [29]. The most difficult point in the in vitro differentiation strategies is to reach the equilibrium between proliferation of precursors needed to reach the adequate mass, and the maturation process to a postmitotic cell. In general it could be accepted that signals that promote proliferation do not render mature cells. In our first published assay we were fortunate enough to obtain cells that still maintain a high proliferation rate and some insulin content [33]. With these cells we assayed different maturation strategies (sodium butyrate, mytomicin C, nicotinamide, low and high glucose, etc.). The best results were obtained by exposing proliferating cell cultures to 10 mm nicotinamide + 25 mm glucose for 2 weeks plus 10 mm nicotinamide + 5 mm glucose for 5 days. This protocol rendered cells with a high insulin content that respond to physiological secretagogues [33]. When these insulin-containing cells were transplanted into streptozotocin-diabetic mice blood glucose was normalized within 1 week and body weight restored in 4 weeks [33]. Long-term follow-up of these animals demonstrate that glucose remains normal after 40 weeks [34]. Furthermore, immunohistochemistry showed insulin-positive cells 4 moths after engraftment [34]. FUTURE TRENDS In addition to diabetes, clinical targets of cell therapy include neurodegenerative disorders (Alzheimer’s
and Parkinson’s diseases), spinal cord injury, cardiovascular diseases, etc. However, before the transplantation of ES-derived cells to humans can be accomplished, several problems have to be solved: cells used in transplantation have to be autologous or immunologically masked, good cell lineage selection procedures have to be developed, the cell population has to be homogeneous with a stable phenotype and, finally, in vitro differentiation and cell maturation strategies have to be developed in order to obtain both a sufficient cell mass and mature post-mitotic cells lacking tumorigenicity. Some recent reports suggest that the functional plasticity of somatic tissue-derived stem cells may be greater than expected. A list of transdifferentiation events, even between cell types belonging to distinct embryonic cell layers, is shown in table II. Then, in addition to using the natural precursor (pancreatic duct cell), which has a limited proliferative capacity, somatic stem cells with a pluripotential capacity and high proliferation rate may be found. Recent and unpublished results from our group show that cells isolated from the human intestinal epithelia, when appropriately cultured, express markers of human embryonic stem cells and may in vitro differentiate into myocytes and cells that express markers of the three embryonic germ layers. Using this approach will not only circumvent the ethical dilemmas surrounding research on embryonic and foetal stem cells, but has an additional advantage: they may be obtained from the same patient, thus allowing autotransplantation. The experience of living with diabetes, either as a patient or as parent of a child with diabetes, represents a great burden both in economic and psychological terms. Despite the considerable pressure to find a definitive cure for this disease, we have to be
Stem cells and diabetes
aware that insulin has been used successfully to treat type 1 diabetic patients for the last 80 years. ACKNOWLEDGEMENTS These studies have been partially supported by grants from the Secretaría de Estado de Universidades e Investigación (PM99-0142); Fundació MaratóTV3 (99-1210); 2000 EASD/Eli Lilly Research Fellowship; Juvenile Diabetes Foundation (JDFI 1-2000575) and Fundación Salud 2000. Technical support of E. Fuster and N. Illera is greatly acknowledged. REFERENCES 1 Alison MR, Poulson R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, et al. Hepatocytes from non-hepatic adult stem cells. Nature 2000 ; 406 : 257. 2 Andrews PW, Damjanov I, Simon D, Banting GS, Carlin C, Dracopoli NC, et al. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Lab Invest 1984 ; 50 : 147-62. 3 Bjornson CRR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999 ; 283 : 534-7. 4 Blau HM, Baltimore D. Differentiation requires continuous regulation. J Cell Biol 1991 ; 112 : 781-3. 5 Boeuf H, Hauss C, Graeve FD, Baran N, Kedinger C. Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells. J Cell Biol 1997 ; 138 : 1207-17. 6 Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 2000 ; 97 : 7999-8004. 7 Bouwens L, De Blay E. Islet morphogenesis and stem cell markers in rat pancreas. J Histochem Cytochem 1960 ; 44 : 947-51. 8 Brazelton TR, Rossi FMV, Keshet GI, Blau H. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000 ; 290 : 1775-9. 9 Brendel M, Hering B, Schulz A, Bretzel R. International Islet Transplant Registry report. Giessen, Germany: University of Giesen 1–20; 1999. 10 Clarke DL, Johansson CB, Wilbertz J, Veress B, Nilsson E, Karlström H, et al. Generalized potential of adult neural stem cells. Science 2000 ; 288 : 1660-3. 11 Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, AlvarezBuylla A. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999 ; 97 : 703-16. 12 Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, et al. Muscle regeneration by bone marrowderived myogenic progenitors. Science 1998 ; 279 : 1528-30. 13 Fuhrmann-Benzakein E, García-Gabay I, Pepper MS, Vasalli JD, Herrera PL. Inducible and irreversible control of gene expression using a single transgene. Nucleic Acids Res 2000 ; 28 : E99. 14 Gage FH. Mammalian neural stem cells. Science 2000 ; 287 : 1433-8. 15 Galli R, Borello U, Gritti A, Minasi MG, Bjornson C, Coletta M, et al. Skeletal myogenic potential of human and mouse neural stem cells. Nat Neurosci 2000 ; 3 : 986-91. 16 Herrera PL. Adult insulin- and glucagons-producing cells differentiate from two independent cell lineages. Development 2000 ; 127 : 2317-22.
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17 Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci U S A 1999 ; 96 : 14482-6. 18 Johansson CB, Momma S, Clarke DL, Risling M, Lendhal U, Frisen J. Identification of a neural stem cell in the adult central nervous system. Cell 1999 ; 96 : 25-34. 19 Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 1996 ; 98 : 216-24. 20 Li M, Pevny L, Lovell-Badge R, Smith A. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr Biol 1998 ; 8 : 971-4. 21 Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood into brain: bearing neuronal antigens generated in vivo from bone marrow. Science 2000 ; 290 : 1779-82. 22 Niwa H, Burton T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998 ; 12 : 2048-60. 23 Niwa H, Miyazaki JI, Smith AG. Quantitative expression of OCT3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000 ; 24 : 372-6. 24 Pera MF, Cooper S, Mills J, Parrington JM. Isolation and characterization of a multipotent clone of human embryonal carcinoma-cells. Differentiation 1989 ; 42 : 10-23. 25 Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999 ; 28 : 1168-70. 26 Real FX, Vila MR, Skoudy A, Ramaekers FC, Corominas JM. Intermediate filaments as differentiation markers of exocrine pancreas. II. Expression of cytokeratins’ complex and stratified epithelia in normal pancreas and in pancreas cancer. Int J Cancer 1993 ; 54 : 707-20. 27 Reubinoff BE, Pera MF, Chui-Yee F, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotech 2000 ; 18 : 399-404. 28 Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 1998 ; 95 : 13726-31. 29 Shapiro AMJ, Lakey JRT, Ryan EA, Korbutt GS, Toth E, Warnock GL, et al. Islet transplantation in seven patients with Type 1 Diabetes Mellitus using a glucocorticoid-free immnuosuppressive regimen. N Engl J Med 2000 ; 343 : 230-8. 30 Slack JMW. Stem cells in epithelial tissues. Science 2000 ; 287 : 1431-3. 31 Song SY, Gannon M, Washington MK, Scoggins CR, Meszoely IM, Goldenring JM, et al. Expansion of PDX1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor alpha. Gastroenterology 2000 ; 117 : 1416-26. 32 Soria B, Andreu E, Berná G, Fuentes E, Gil A, León-Quinto T, et al. Engineering pancreatic islets. Pflügers Arch-Eur J Physiol 2000 ; 440 : 1-18. 33 Soria B, Roche E, Berná G, León-Quinto T, Reig JA, Martín F. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000 ; 49 : 157-62. 34 Soria B, Skoudy A, Martín F. From stem cells to B-cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 2001 (in press). 35 Tada M, Tada T, Lefebvre L, Barton SC, Surani MA. Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells. EMBO J 1997 ; 16 : 6510-20. 36 The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes in the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Eng J Med 1993 ; 329 : 977-86.
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37 Thompson S, Stern PM, Webb M, Walsh FS, Engstrom W, Evans EP, et al. Cloned human teratoma cells differentiate into neuron-like cells and other cell types in retinoic acid. J Cell Sci 1984 ; 72 : 37-64. 38 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998 ; 282 : 1145-7. 39 Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, McInnes RR, et al. Retinal stem cells in the adult mammalian eye. Science 2000 ; 287 : 2032-6. 40 Watanabe T, Yonemura Y, Yonekura H, Suzuki Y, Miyashita H, Sugiyama K, et al. Pancreatic-beta-cell replication and amelioration of surgical diabetes by Reg protein. Proc Natl Acad Sci U S A 1994 ; 91 : 3589-92.
41 Watt FM. Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond 1998 ; 353 : 831-7. 42 Watt FM, Hogan BLM. Out of eden: stem cells and their niches. Science 2000 ; 287 : 1427-30. 43 Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000 ; 100 : 157-68. 44 Williams RL, Hilton DJ, Pease S, Wilson TA, Stewart CL, Gearing DP, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988 ; 336 : 684-7. 45 Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosc Res 2000 ; 61 : 364-70.
Biomed Pharmacother 2001 ; 55 : 206-12 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0753332201000506/FLA
Stem cells and diabetes G. Berná1, T. León-Quinto1, R. Enseñat-Waser1, E. Montanya2, F. Martín1, B. Soria1* 1 Institute of Bioengineering, University Miguel Hernández, Campus de San Juan, E-03550, Alicante, Spain; 2Laboratory of Diabetes and Experimental Endocrinology, Endocrine Unit (13-2), CSUB-Hospital of Bellvitge, University of Barcelona, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Barcelona, Spain
(Received 22 January 2001; accepted 29 January 2001)
Summary – Diabetes mellitus is a metabolic disorder affecting 2–5% of the population. Transplantation of isolated islets of Langerhans from donor pancreata could be a cure for diabetes; however, such an approach is limited by the scarcity of the transplantation material and the long-term side effects of immunosuppressive therapy. These problems may be overcome by using a renewable source of cells, such as islet cells derived from stem cells. Stem cells are defined as clonogenic cells capable of both self-renewal and multilineage differentiation. This mean that these cells can be expanded in vivo or in vitro and differentiated to produce the desired cell type. There exist several sources of stem cells that have been demonstrated to give rise to pluripotent cell lines: 1) embryonic stem cells; 2) embryonic germ cells; 3) embryonic carcinoma cells; and 4) adult stem cells. By using in vitro differentiation and selection protocols, embryonic stem cells can be guided into specific cell lineages and selected by applying genetic selection when a marker gene is expressed. Recently, differentiation and cell selection protocols have been used to generate embryonic stem cell-derived insulin-secreting cells that normalise blood glucose when transplanted into diabetic animals. Some recent reports suggest that functional plasticity of adult stem cells may be greater than expected. The use of adult stem cells will circumvent the ethical dilemma surrounding embryonic stem cells and will allow autotransplantation. These investigations have increased the expectations that cell therapy could be one of the solutions to diabetes. © 2001 Éditions scientifiques et médicales Elsevier SAS diabetes / stem cells / transplantation
Diabetes mellitus is a heterogeneous metabolic disorder affecting 2–5% of the adult population in developed countries. Worldwide prevalence figures give an estimate of 130 million people in 2000 and 300 million in 2025. Diabetes mellitus can be classified broadly into two groups: the insulin-dependent type (IDDM or type 1), in which the treatment mainly relies on the self-injection of insulin several times daily, and the non-insulin-dependent type (NIDDM or type 2). Despite the distinct etiology et pathophysi*Correspondence and reprints. E-mail address: [email protected] (B. Soria).
ology both types may result in late complications (nephropathy, retinopathy, neuropathy, etc.). The Diabetes Control and Complications Trial [36] has shown that tight control of blood glucose can delay and diminish the progression of long-term complications; however, intensive insulin therapy (5–6 injections daily) and almost permanent blood glucose control requires highly motivated patients and does not liberate them from insulin therapy. Transplantation of insulin-producing cells isolated from donor pancreata could be a cure for type 1 and some cases of type 2 diabetes. In recent years, islet transplantation failed to materialize the hope for long-term
Stem cells and diabetes
freedom of exogenous insulin. Less than 10% of the almost 300 islet allografts transplanted since 1990 resulted in insulin independence for periods of more than 12 months [9]. A recent report by Shapiro et al. has demonstrated that using a glucocorticoid-free immnunosuppresive therapy combined with the infusion of an adequate fresh islet mass resulted in insulin independence and good metabolic control for periods of more that 12 months in seven type 1 diabetic patients [29]. However, the therapeutic potential of this approach is limited by the scarcity of transplantation material and the long-term side effects of immunosuppressive therapy. On the other hand, the potential risk of infection by animal endogenous viruses has prevented so far the therapeutic uses of islet xenografts. These problems may be overcome by deriving islet cells from stem cells. This review will concentrate on the pathway from progenitors to insulin-containing cells, either early progenitors (embryonic stem cells, ESC) or near progenitors (ductal cells). STEM CELLS: CONCEPT, PROPERTIES AND TYPES Stem cells are defined as clonogenic cells capable of both self-renewal and multilineage differentiation [42]. However, self-renewal in itself does not define stem cells and these may be non-dividing or slowlydividing cells. Some adult stem cells are commonly defined as those that can be activated to undergo proliferation upon tissue injury to achieve functional restitution. Between the stem cell and its terminally differentiated progeny there is usually an intermediate population of committed progenitors with limited proliferative capacity and restricted differentiation potential; these are the cells that display greatest proliferative activity under physiological and pathophysiological circumstances. The mechanisms of differentiation and the intermediate states can vary in different species and/or tissues, but all of them share these two properties: the capability to produce identical daughter cells (‘self-renewal’), and the possibility to produce daughters that are fated to differentiate [42]. Whilst the former means that they can be expanded, the latter indicates that they can differentiate to produce the desired cell type, either in vivo or in vitro. In terms of potentiality, they may be classified as totipotential (giving rise to any cell type including the trophoblasts of the placenta), pluripo-
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tential (when they may differentiate into any cell type including the germ line) and unipotential. Unipotential stem cells are cell lineage progenitors committed to differentiate into a particular cell type [30]. These properties offer tremendous potential for clinical application, serving as an unlimited source of cells for transplantation and tissue regeneration therapies, as well as intensive drug and toxic screening as an alternative method for animal assays. The earliest stem cell in ontogeny is totipotent (from zygote to the inner cell mass of the blastocyst); soon thereafter, totipotent cells give rise to somatic stem, progenitor cells and primitive germ-line stem cells [43]. Very little is known of the stages between somatic stem cells and the emergence of tissue- or organspecific stem cells in the neurula stage. At this stage haematopoietic stem cells appear. Since these are the best known stem cells, most of the concepts have been developed after them, though it is not clear that direct extrapolations can be made to other stem cell types. In addition, stem cells are powerful tools for studying the control of in vitro cell differentiation. A number of properties besides self-renewal and differentiation potential have been ascribed to stem cells, including the ability to undergo asymmetric cell divisions, to exist in a mitotically quiescent form and to clonally regenerate [42]. However, maintenance and/or loss of the ‘stemness’ properties depend on cell autonomous regulators (transcription factors and internal clocks) modulated by external signals (secreted factors, cell-to-cell and cell-to-matrix interactions) [42]. Differentiation of ESC (obtained from the inner cell mass of the blastocyst) occurs in vitro when the growth factor signals for self-renewal are not sufficient in the culture medium. The self-renewal of undifferentiated mouse ESC depends on an exogenous supply of the cytokine leukaemia inhibitory factor (LIF) [44], which binds to LIF receptor (LIFR) and gp130 signalling complex, thereby activating STAT3 and the receptor-associated Janus kinase (JAK) [5, 22]. Together with the activation of the LIF-signalling pathway, the POU transcription factor Oct-3/4 acts as a main regulator of pluripotency that controls lineage commitment [23]. On the other hand, the exact nature of these signals is unknown for human ESC, which expresses Oct-4 but does not rely on an exogenous supply of LIF to sustain their proliferation as undifferentiated stem cells [27, 38]. In this regard, human ESC underwent low levels of
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spontaneous differentiation during routine passaging and this process was accelerated by growing cultures at high density for 4 to 7 weeks [27, 38]. Finally, spontaneous differentiation of human ESC into cell types originating from each of the three embryonic germ layers also occurs in vivo when grafting them into immunocompromised severe combined immunodeficient (SCID) mice [27, 38]. SOURCES OF STEM CELLS Pluripotent and totipotent cell lines Three main primary sources of human tissue have been demonstrated to give rise to pluripotent cell lines: 1) ESC derived from the inner cell mass of human blastocyst stage embryos [27, 38]; 2) embryonic germ cells (EGC) derived from the gonadal region of 5- to 9-week-old aborted human foetuses [28]; and 3) embryonic carcinoma cells (ECC) derived from teratocarcinoma tissue [2] (table I). A fundamental difference between these cell lines is that whilst ESC and EGC lines are genetically normal and diploid, ECC lines are transformed and are commonly aneuploid. They also differ in their cell surface marker expression, cell culture conditions, pluripotentiality and clinical utility. Thus whereas ECC lines have been of use in cell differentiation studies of early human development [2, 24, 37], ESC and EGC may also serve as a source of desired cells, or even as enucleated recipients of nuclei from patient cells instead of enucleated oocytes [35]. This procedure will lead into ‘customised’ pluripotent stem cells, which will overcome the present difficulties related to immune compatibility and graft rejection, with the subsequent use of immunosuppressive drugs. Adult stem cell lines Certain tissues maintain the capability to regenerate themselves during an entire lifetime due to the presence of stem cells. Skin, intestinal epithelia or blood cells display a high rate of cell renewal, including both cell proliferation and death, in the normal adult, even in the absence of injury. Other tissues such as liver or muscle may regenerate when injured. Finally, it is generally thought that terminally differentiated cells have suspended their differentiation possibilities and maintain a fixed phenotype with negligible
Table I. Stem cells that may potentially be used to derive pancreatic B-cells. Totipotential stem cells Embryonic stem cells Embryonic germ cells Embryonic carcinoma cells Multipotential stem cells Intestinal epithelia cells Bone marrow stroma cells Others Committed stem cells Fetal pancreatic stem cells Pancreatic duct cells Others
division potential. For many years this has been the accepted paradigm. However, recently some of the accepted paradigms have fallen into discredit. Several studies have shown that adult stem cells can be derived from tissues thought to lack regenerative capacity. Neural [14], retina [39] and bone marrow [45] are examples of tissues that have been incorporated into the category of those that possess stem cells. Furthermore, some of these adult stem cells are not irreversibly committed to a particular fate and appear to have a much broader capacity of differentiation than was previously recognised. For example, two groups have recently described the presence of multipotential adult stem cells from brain tissue of adult mice [11, 18]. These two groups have demonstrated that cells from different origins (ependymal cells [18] or astrocytes [11]) may give rise to distinct neural cells. In addition, some studies indicate that neural stem cells can produce a variety of blood cell types [3] or that stem cells isolated from muscle are capable of forming haematopoietic cell lineages [17], and haematopoietic stem cells have been shown to give rise to a variety of tissues [25]. Moreover, Clarke et al. have recently demonstrated that adult neural stem cells can contribute to the formation of chimeric chicks and mice, indicating that adult neural stem cells have a very broad developmental capacity [10]. This violation of the accepted paradigm does not preclude the search for adequate grounds that the notion which states “differentiation requires continuous regulation” [4] is fully supported. As in the case of ESC, the signals that control the maintenance and/or loss of the somatic stem cell phenotype and fate are only partially known. In addition, very little is known about molecular markers of the stem cell phenotype
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in adult tissues. In the case of epidermal stem cells, high levels of β1 integrin (an integral membrane protein responsible for cell adhesion to the extracellular matrix) are characteristic of cells with stem cell properties and β1-mediated cell adhesion suppresses the onset of terminal differentiation [41, 42]. IN-VITRO DIFFERENTIATION AND CELLLINEAGE SELECTION STRATEGIES Embryonic stem cells can be propagated indefinitely in vitro and, under certain conditions, induced to differentiate into several lineages such as cardiomyocytes (mesoderm derivative) [19], or neural precursors (ectoderm derivative) [20]. In contrast, in vitro differentiation into endoderm-derived cells, such as pancreatic B-cells, is not spontaneously produced. The sequence, combination and amplitude of the signalling mechanisms driving a stem cell into a committed pathway are not well known and are probably governed by biochemical and biophysical factors. However, the combination of cell density, growth factors and cell polarization may result in an enrichment of certain cell populations which express markers of endoderm-derived cells such as amylase, elastase, carboxypeptidase, PDX-1, insulin, glucagons, etc.). Subsequent cell lineage selection may result in highly pure cell types. Using an ‘intelligent’ transgene (figure 1, GB-1) that couples the insulin gene with the neomycin-resistant gene has made possible the selection of insulin-producing cells based on their antibiotic resistance. This cell lineage selection strategy allows multiple variants, such as using the regulatory region (figure 1, GB-2), the promoter region of the gene encoding the transcription factor PDX-1 (figure 1, GB-3) or the pancreatic polypeptide promoter (figure 1, GB-4). Both PDX-1 and PP positive cells have been postulated as B-cell precursors [16]. Furthermore, any of these precursors can be coupled to the GFP gene and use the protein fluorescence to identify and separate the desired cell line. Finally, flanking the construct with the recombinase inducible gene (Cre) allows the removal of the chimeric DNA construct after the selection of the cell type [13]. Identification of the B-cell precursor together with a more detailed knowledge of the signals that drive later steps in B-cell differentiation could, in theory, be used for in vitro growth and differentiate precursor cells taken either from aborted foetuses or pan-
Figure 1. Structure of the transgenes used in cell-lineage selection strategies. GB-1: human insulin gene-β-geo/pGK-hygror construction; GB-2: human insulin promoter-β-geo/pGK-hygror; GB-3: PDX-1 promoter-β-geo/pGK-hygror; GB-4: PP promoter-β-geo/pGKhygror.
creatic ducts. The exact nature of the pancreatic stem cell is still uncertain; several markers have been proposed to identify stem cells, including PDX-1 [31], BCL-2 [7] and cytokeratins 5 and 14 [26]. Furthermore, is not known whether B-cell precursors in the adult are distinct from foetal B-cell precursors. Replication is considered to be the principal mechanism in increasing B-cell mass after birth [40]. Both replication of preexisting B-cells and differentiation from pancreatic duct epithelium can take place in the adult life and offer a possibility for the expansion of the B-cell mass [32]. Recently, human pancreatic duct cells have been grown and induced to differentiate in vitro [6]. The recipe includes adding keratinocyte growth factor, nicotinamide and ITS medium (insulin + transferrin + selenium) during 1–2 weeks to promote proliferation, followed by coating with a murine basement membrane preparation (Matrigelt). The use of adult donor ductal cells avoids the controversy of using foetal or embryonic stem cells by using a material which, in principle, is more committed to
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Table II. Transdifferentiation events observed in the literature. From
To
Ref.
Neural cells Muscle cells Neural cells Bone stroma cells
Blood cells Blood cells Chimeric mouse Neural cells
Neural cells Bone stroma cells Bone stroma cells
Muscle cells Muscle cells Liver cells
Bjornson, 1999 [3] Jackson, 1999 [17] Clarke, 2000 [10] Woodbury, 2000 [45] Brazelton, 2000 [8] Mezey, 2000 [21] Galli, 2000 [15] Ferrari, 1998 [12] Alison, 2000 [1]
differentiate into B-cells. Unfortunately, the expansion capability is more limited and restricts its clinical utility since the total islet mass transplanted is a determinant factor for reaching insulin independence. Based in fresh islet transplantation an islet mass of approximately 12,000 islet equivalent/kg of body weight is required [29]. The most difficult point in the in vitro differentiation strategies is to reach the equilibrium between proliferation of precursors needed to reach the adequate mass, and the maturation process to a postmitotic cell. In general it could be accepted that signals that promote proliferation do not render mature cells. In our first published assay we were fortunate enough to obtain cells that still maintain a high proliferation rate and some insulin content [33]. With these cells we assayed different maturation strategies (sodium butyrate, mytomicin C, nicotinamide, low and high glucose, etc.). The best results were obtained by exposing proliferating cell cultures to 10 mm nicotinamide + 25 mm glucose for 2 weeks plus 10 mm nicotinamide + 5 mm glucose for 5 days. This protocol rendered cells with a high insulin content that respond to physiological secretagogues [33]. When these insulin-containing cells were transplanted into streptozotocin-diabetic mice blood glucose was normalized within 1 week and body weight restored in 4 weeks [33]. Long-term follow-up of these animals demonstrate that glucose remains normal after 40 weeks [34]. Furthermore, immunohistochemistry showed insulin-positive cells 4 moths after engraftment [34]. FUTURE TRENDS In addition to diabetes, clinical targets of cell therapy include neurodegenerative disorders (Alzheimer’s
and Parkinson’s diseases), spinal cord injury, cardiovascular diseases, etc. However, before the transplantation of ES-derived cells to humans can be accomplished, several problems have to be solved: cells used in transplantation have to be autologous or immunologically masked, good cell lineage selection procedures have to be developed, the cell population has to be homogeneous with a stable phenotype and, finally, in vitro differentiation and cell maturation strategies have to be developed in order to obtain both a sufficient cell mass and mature post-mitotic cells lacking tumorigenicity. Some recent reports suggest that the functional plasticity of somatic tissue-derived stem cells may be greater than expected. A list of transdifferentiation events, even between cell types belonging to distinct embryonic cell layers, is shown in table II. Then, in addition to using the natural precursor (pancreatic duct cell), which has a limited proliferative capacity, somatic stem cells with a pluripotential capacity and high proliferation rate may be found. Recent and unpublished results from our group show that cells isolated from the human intestinal epithelia, when appropriately cultured, express markers of human embryonic stem cells and may in vitro differentiate into myocytes and cells that express markers of the three embryonic germ layers. Using this approach will not only circumvent the ethical dilemmas surrounding research on embryonic and foetal stem cells, but has an additional advantage: they may be obtained from the same patient, thus allowing autotransplantation. The experience of living with diabetes, either as a patient or as parent of a child with diabetes, represents a great burden both in economic and psychological terms. Despite the considerable pressure to find a definitive cure for this disease, we have to be
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aware that insulin has been used successfully to treat type 1 diabetic patients for the last 80 years. ACKNOWLEDGEMENTS These studies have been partially supported by grants from the Secretaría de Estado de Universidades e Investigación (PM99-0142); Fundació MaratóTV3 (99-1210); 2000 EASD/Eli Lilly Research Fellowship; Juvenile Diabetes Foundation (JDFI 1-2000575) and Fundación Salud 2000. Technical support of E. Fuster and N. Illera is greatly acknowledged. REFERENCES 1 Alison MR, Poulson R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, et al. Hepatocytes from non-hepatic adult stem cells. Nature 2000 ; 406 : 257. 2 Andrews PW, Damjanov I, Simon D, Banting GS, Carlin C, Dracopoli NC, et al. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Lab Invest 1984 ; 50 : 147-62. 3 Bjornson CRR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999 ; 283 : 534-7. 4 Blau HM, Baltimore D. Differentiation requires continuous regulation. J Cell Biol 1991 ; 112 : 781-3. 5 Boeuf H, Hauss C, Graeve FD, Baran N, Kedinger C. Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells. J Cell Biol 1997 ; 138 : 1207-17. 6 Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 2000 ; 97 : 7999-8004. 7 Bouwens L, De Blay E. Islet morphogenesis and stem cell markers in rat pancreas. J Histochem Cytochem 1960 ; 44 : 947-51. 8 Brazelton TR, Rossi FMV, Keshet GI, Blau H. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000 ; 290 : 1775-9. 9 Brendel M, Hering B, Schulz A, Bretzel R. International Islet Transplant Registry report. Giessen, Germany: University of Giesen 1–20; 1999. 10 Clarke DL, Johansson CB, Wilbertz J, Veress B, Nilsson E, Karlström H, et al. Generalized potential of adult neural stem cells. Science 2000 ; 288 : 1660-3. 11 Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, AlvarezBuylla A. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999 ; 97 : 703-16. 12 Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, et al. Muscle regeneration by bone marrowderived myogenic progenitors. Science 1998 ; 279 : 1528-30. 13 Fuhrmann-Benzakein E, García-Gabay I, Pepper MS, Vasalli JD, Herrera PL. Inducible and irreversible control of gene expression using a single transgene. Nucleic Acids Res 2000 ; 28 : E99. 14 Gage FH. Mammalian neural stem cells. Science 2000 ; 287 : 1433-8. 15 Galli R, Borello U, Gritti A, Minasi MG, Bjornson C, Coletta M, et al. Skeletal myogenic potential of human and mouse neural stem cells. Nat Neurosci 2000 ; 3 : 986-91. 16 Herrera PL. Adult insulin- and glucagons-producing cells differentiate from two independent cell lineages. Development 2000 ; 127 : 2317-22.
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