Stem Cells in Endocrinology - part 6 ppsx

CONTENTS INTRODUCTION TRANSCRIPTION FACTOR-DIRECTED DIFFERENTIATION OF NONENDOCRINE CELL TYPES USE OF TRANSCRIPTION FACTORS TO DIRECT DIFFERENTIATION ALONG AN ENDOCRINE CELL LINEAGE in d

28 Pictet R, Rutter WJ Development of the embryonic pancreas In: Steiner DF, Frenkel N, eds Handbook of Physiology, Section 7 Washington, DC, American Physiological Society, 1972,

pp 25–66.

29 Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE A second pathway for regeneration of adult exocrine and endocrine pancreas: a possible recapitulation of embryonic development Diabetes 1993;42:1715–1720.

30 Rooman I, Lardon J, Bowens L Gastrin stimulates β cell neogenesis and increases islet mass from transdifferentiated but not from normal exocrine pancreas tissue Diabetes 2002;51:686–690.

31 Rooman I, Hereman Y, Heimberg H, Bowens L Modulation of rat pancreatic acinoductal transdifferentiation and expression of Pdx-1 in vitro Diabetologia 2000;43:907–914.

32 Bonner-Weir S, Taneja M, Weir G, et al In vitro cultivation of human islets from expanded ductal tissue Proc Natl Acad Sci USA 2000;97:7999–8005.

33 Gao R, Ustinov J, Pulkkinen MA, Lundin K, Korsgren O, Otonkoski T Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancre- atic cell culture Diabetes 2003;52:2007–2015.

34 Zulewski H,Abraham EJ, Gerlach MJ, et al Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex-vivo into pancreatic endocrine, exocrine and hepatic phenotypes Diabetes 2001;50:521–533.

35 Alpert S, Hanahan D, Teitelman G Hybrid insulin genes reveal a developmental lineage for pancreatic endocrine cells and imply a relationship with neurons Cell 1988;53:295–308.

36 Gittes GK, Rutter WJ Onset of cell-specific gene expression in the developing mouse creas Proc Natl Acad Sci USA 1992;89:1128–1132.

pan-37 Herrera PL, Huarte J, Sanvito F, Meda P, Orci L, Vassalli JD Embryogenesis of the murine endocrine pancreas; early expression of the pancreatic polypeptide gene Development 1991;113:1257–1265.

38 Golosow N, Grobstein C Epitheliomesenchymal interaction in pancreatic morphogenesis Dev Biol 1962;4:242–255.

39 Wessels NK, Cohen JH Early pancreas organogenesis: morphogenesis, tissue interactions, and mass effects Dev Biol 1967;15:237–270.

40 Upchurch B, Aponte GW, Leiter AB Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY producing progenitor Devel- opment 1994;120:245–252.

41 Gannon M, Wright CVE Endodermal patterning and organogenesis In: Mood S, ed Cell Lineage and Fate Determination New York, Academic Press, 1999, pp 583–615.

42 Guz Y, Montminy MR, Stein R, et al Expression of murine stf-1, a putative insulin gene transcription factor, in β cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny Development 1995;121:11–18.

43 Offield MF, Jetton TL, Labosky PA, et al PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum Development 1996;122:983–995.

44 Naya FJ, Stellrecht CM, Tsai MJ Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor Genes Dev 1995;9:1009–1019.

45 Naya FJ, Huang HP, Qui Y, et al Diabetes, defective pancreatic morphogenesis, and mal enteroendocrine differentiation in BETA2-NeuroD-deficient mice Genes Dev 1997;11: 2323–2334.

abnor-46 Herrera PL Adult insulin and glucagon-producing cells differentiate from two independent lineages Development 2000;127:317–2322.

47 Pang K, Mukonoweshuro C, Wong GC Beta cells arise from glucose transporter type 2)-expressing epithelial cells of the developing rat pancreas Proc Natl Acad Sci USA 1994;91:9559–9563.

2(Glut-48 Teitelman G, Alpert S, Polak JM, Martinez A, Hanahan D Precursor cells of mouse endocrine pancreas coexpress insulin,glucagon,and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide Development 1993;118:1031–1039.

49 Vincent M, Guz Y, Rozenberg M, et al Abrogation of protein convertase 2 (PC-2) activity results in delayed islet cell differentiation and maturation, increase in alpha cell proliferation and islet neogenesis Endocrinology 2003;144:4061–4069.

50 Fernandes A, King LC, Guz Y, Stein R, Wright CVE, Teitelman G Differentiation of new insulin producing cells is induced by injury in adult pancreatic islets Endocrinology 1997;138:1750–1762.

51 Guz Y, Nasir I, Teitelman G Regeneration of pancreatic β cells from intra-islet precursor cells

in an experimental model of diabetes Endocrinology 2001;142:4956–4969.

52 Like AA, Rossini AA Streptozotocin-induced pancreatic insulinitis: new model of diabetes mellitus Science 1976;193:415–418.

53 Rodrigues B, Poucheret P, Batell ML, McNeill JH In: McNeill JH, ed induced diabetes: induction, mechanisms(s), and dose dependency Experimental Models

Streptozotocin-of Diabetes Boca Raton, FL, CRC Press, 1999, pp 3–14.

54 Guz Y, Torres A, Teitelman G Detrimental effect of protracted hyperglycaemia on beta-cell neogenesis in a mouse murine model of diabetes Diabetologia 2002;45:1689–1696.

55 Leiter EH, Gerling IC, Flynn JC Spontaneous insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic(NOD) mice: comparison with experimentally induced IDDM In: McNeill

JH, ed Experimental Models of Diabetes Boca Raton, FL, CRC Press, 1999, pp 257–294.

56 Reddy S, Young M, Poole CA, JM Ross Loss of glucose transporter-2 precedes insulin loss

in the non-obese diabetic and the low-dose streptozotocin mouse models: a comparative immunohistochemical study by light and confocal microscopy Gen Comp Endocrinol 1998;111:9–19.

57 Sorenson RL, Brejle TC Adaptation of islets of Langerhans to pregnancy: β cell growth, enhanced insulin secretion and the role of lactogenic hormones Horm Metab Res 1996;29:301–307.

58 Nielsen JH, Galsgaard ED, Moldrup A, et al Regulation of β cell mass by hormones and growth factors Diabetes 2001;50(Suppl 1):S25–S29.

59 Wang J, Webb G, Cao Y, Steiner DF Contrasting patterns of expression of transcription factors in pancreatic alpha and beta cells Proc Natl Acad Sci USA 2003;100:12660–12665.

From: Contemporary Endocrinology: Stem Cells in Endocrinology

Edited by: L B Lester © Humana Press Inc., Totowa, NJ

Differentiation of Stem Cells

Along an Endocrine Lineage

William L Lowe, Jr.

CONTENTS

INTRODUCTION

TRANSCRIPTION FACTOR-DIRECTED DIFFERENTIATION

OF NONENDOCRINE CELL TYPES

USE OF TRANSCRIPTION FACTORS TO DIRECT DIFFERENTIATION

ALONG AN ENDOCRINE CELL LINEAGE

in diabetes, complications of long-term overreplacement of hydrocortisone orinadequate hydrocortisone replacement during times of stress) Thus cell replace-ment therapy capable of restoring endocrine function similar to that of the nativegland would represent a major therapeutic advance To that end, the differentia-tion of stem cells to generate new endocrine cells offers great potential

As described in other chapters, a number of different approaches can beemployed to differentiate embryonic or other stem cells along a specific lin-eage One approach that has been employed is forced differentiation This can

be accomplished by expressing a gene important for cell lineage determination

to direct stem cell differentiation along a specific pathway Typically, thesegenes initiate a hierarchical cascade of gene expression that ultimately results incell differentiation Beyond providing a means to develop cells capable of beingused for cell replacement therapy, this approach of using transcription factorexpression to direct stem cell differentiation also provides important insight intothe genetic programs directed by different transcription factors and the develop-mental program of different cell types This chapter will describe how this ap-proach has been used to develop partially or fully differentiated cells capable ofreplacing cell function

2 TRANSCRIPTION FACTOR-DIRECTED DIFFERENTIATION

OF NONENDOCRINE CELL TYPES

Multiple approaches have been used to successfully transfer DNA into stemcells and permit expression of specific genes (e.g., stable transfection of DNAafter electroporation, use of adenoviral or lentiviral vectors) To date, the approach

of directed differentiation via transcription factor expression in stem cells has beenused to greatest effect to generate nonendocrine cells Thus, a few examples ofdirected differentiation of stem cells into nonendocrine cells are described

2.1 Hematopoietic Cells

Removing embryonic stem (ES) cells from feeder cells or leukemia inhibitoryfactor, both of which inhibit ES cell differentiation, and placing them on anonadherent surface results in the formation of clusters of cells referred to asembryoid bodies Within embryoid bodies, ES cells spontaneously differentiateand generate cells from all three germ layers (i.e., mesoderm, ectoderm, andendoderm) Among the cell types formed in embryoid bodies are blood elements.However, the differentiation of blood elements in embryoid bodies appears torecapitulate primitive hematopoiesis, which occurs in the yolk sac, and notdefinitive hematopoiesis, which is mediated by definitive hematopoietic stem

cells and persists throughout life (reviewed in ref 1) Given the inability to

generate definitive hematopoietic stem cells from ES cells, long-term stableengraftment of ES-derived hematopoietic cells in bone marrow after transplan-tation into irradiated recipients has not been accomplished To address the prob-lem of generating definitive hematopoietic stem cells, screens to define factorsimportant for hematopoietic stem cell development have been undertaken Fromthese screens, strategies have been developed to generate transplantable ES cell-derived hematopoietic stem cells capable of engrafting in the bone marrow ofirradiated mice

Among the factors identified in these screens was the transcription factor

HoxB4 (1) HoxB4 is a homeobox transcription factor and a member of a family

of genes that are transcribed from four clusters referred to as HoxA, HoxB, HoxC, and HoxD (2) Several members of this large family of genes, including HoxB4,

are important for hematopoietic lineage commitment A second factor identified

in the screens was the transcription factor Stat5 (3) Stat5 is a member of a family

of transcription factors that are present in the cytoplasm and form homo- or

heterodimers following tyrosine phosphorylation (4) The phosphorylated dimers

translocate to the nucleus where they mediate a program of gene expression TheStats are activated by a variety of cytokines and other peptides, including thosethat are important for hematopoiesis Stat5 is downstream of the Bcr/Abloncogene, which is important in the pathogenesis of chronic myelogenous leu-

kemia and regulates definitive hematopoietic stem cells (3,5).

To determine the impact of either HoxB4 or Stat5 expression on the tiation of ES cells, ES cells capable of doxycycline-inducible expression of one

differen-of the two transcription factors were developed (3,6) In the case differen-of Stat5, a

mutant form of the protein which is constitutively active was expressed In bothcases, the transcription factors were expressed from day 4 to day 6 of cell differ-entiation in embryoid bodies Expression of both transcription factors enhancedthe formation of hematopoietic colony-forming cells Importantly, subsequentculturing of the cells on stromal cells in the presence of cytokines and doxycy-cline generated hematopoietic blast cells Transplantation of the HoxB4- andStat5-induced ES-derived hematopoietic cells into irradiated syngeneic micehad different outcomes HoxB4-induced cells were able to home to the bonemarrow, contribute to myeloid and lymphoid lineages, and be represented in the

hematopoietic stem cell pool (6) Stat5-expressing cells were able to engraft only

in the presence of the continued induced expression of Stat5, and, even underthese conditions, their contribution to hematopoietic lineages was lost after 8

weeks (1) Despite the more limited potential of these cells, Stat5 expression

clearly augmented commitment of ES cells to a hematopoietic pathway Thesestudies demonstrate the potential utility of manipulating gene expression as ameans to direct cell differentiation, and, in the case of cells expressing Stat5,demonstrate that activation of an effector of specific signaling pathways was able

to direct ES cell differentiation

2.2 Neural Cells

Several different approaches have been employed to direct or augment thedifferentiation of ES cells into neural cells Among the earliest genes to beexpressed in neuroepithelium during differentiation of neural cells are basichelix–loop–helix transcription factors that are members of the NeuroD/

neurogenin family (7) NeuroD3 is expressed early, followed by expression of

NeuroD1 and NeuroD2 Stable transfection of ES cells with vectors that express

a member of the NeuroD family followed by growth under conditions that

pro-mote ES cell differentiation resulted in differentiation along a neural lineage (8).

Depending on the transcription factor that was expressed, the phenotype of thecells varied Expression of NeuroD3 resulted in primitive-appearing neural cellsthat were bipolar with short, branched processes In contrast, cells expressingNeuroD2 were unipolar with longer processes.

The SOX proteins are a family of transcription factors that contain an

HMG-box DNA binding domain (9) Members of this family, including SOX1, SOX2,

and SOX3, appear to contribute to cell fate decisions in the developing nervous

system (9) SOX1 expression occurs at the time of neural induction, suggesting that it may direct cells toward a neural fate (10) Indeed, in embryonal carcinoma

cells, which, as with ES cells, are capable of differentiating into all three germlayers, treatment with retinoic acid induces neural differentiation and stimulates

SOX1 expression (10) Similarly, expression of a Sox1 cDNA in embryonal

carcinoma cells results in neural differentiation, as reflected by the expression of

neuroepithelial and neuronal markers (10) Importantly, SOX1 was expressed in

an inducible fashion in the embryonal carcinoma cells, and only transient sion of SOX1 was required to induce neural differentiation In this example ofusing a transcription factor to direct differentiation, SOX1 expression was able tosubstitute for a known inductive factor, retinoic acid In other tissues, the geneticprograms responsible for tissue development and cell differentiation are beingelucidated, but the inductive factors that stimulate them remain more obscure.This example suggests that expressing genes that initiate and direct genetic pro-grams stimulated by inductive factors is one approach to direct differentiationalong a specific pathway

expres-In addition to using transcription factor expression to initiate a genetic gram that directs stem cell differentiation, transcription factor expression canalso be used to augment the differentiation of ES cells along a specific pathway.Cells of potential clinical importance are midbrain neurons that secrete dopamine,because they offer a potential therapy for Parkinson’s disease The generation ofthese cells has been accomplished by modifying a previously devised method forthe differentiation of ES cells into neurons Specifically, the proportion of neu-rons capable of producing dopamine was increased by treating cells late in the

pro-differentiation process with fibroblast growth factor 8 and sonic hedgehog (11).

Among the transcription factors induced by treatment with sonic hedgehog and

fibroblast growth factor 8 is nuclear receptor related-1 (Nurr1) (11) To augment

the differentiation of cells into dopamine-secreting neurons, a cDNA-encodingNurr1 was stably and constitutively expressed in ES cells, and the cells were thensubjected to the same differentiation protocol This increased the proportion ofneurons expressing tyrosine hydroxylase, the enzyme responsible for conversion

of tyrosine to dopamine, from approximately 20% to 78% (12) Consistent with

this, these cultures produced greater amounts of dopamine and expressed higherlevels of mRNA encoding proteins important for the development and function

of dopamine neurons Most important, the differentiated Nurr1-expressing cellswere more effective in correcting abnormal behaviors when transplanted into

rodents in which a Parkinson’s disease-like syndrome had been induced (12).

2.3 Endoderm Development

Endocrine glands such as the pancreas and thyroid are derived from derm To date, differentiation of ES cells into cells of endodermal origin hasproven more challenging than differentiation into cells of mesodermal or ecto-dermal origin Among the transcription factors expressed in early endodermlayers from which the pancreas arises are Foxa1 and Foxa2 (previously referred

endo-to as hepaendo-tocyte nuclear facendo-tor 3α [HNF3α] and 3b [HNF3β], respectively) (13–

15) Mice with a null mutation of Foxa2 fail to develop foregut and mid-gut endoderm (16,17) When ES cells overexpressing HNF3β were differentiated in

embryoid bodies, increased expression of genes present in endoderm-derivedtissues, including albumin and the cystic fibrosis transmembrane conductanceregulator, was observed, although genes expressed late in endoderm differentia-tion (e.g., α1-antitrypsin and phosphoenolpyruvate carboxykinase) were expressed

either at low levels or not all (15) Overexpression of HNF3α markedly increased

cystic fibrosis transmembrane conductance regulator expression, but had only a

small effect on albumin expression (15) These studies demonstrate that

expres-sion of specific transcription factors is able to initiate a series of regulatory eventsthat directs differentiation along an endoderm lineage Such an approach mayhold promise for facilitating the differentiation of ES cells into endocrine glands

3 USE OF TRANSCRIPTION FACTORS TO DIRECT DIFFERENTIATION ALONG AN ENDOCRINE CELL LINEAGE

Examples of using transcription factors to direct the differentiation of stemcells along an endocrine lineage are more limited To date, most effort has beendirected toward the development of insulin-secreting cells, although thisapproach has also been used to generate cells capable of steroid hormonesynthesis These efforts are described in the following sections

3.1 Insulin-Secreting Cells

Type 1 diabetes occurs secondary to the autoimmune-mediated destruction ofinsulin-producing β-cells in pancreatic islets In contrast, insulin resistance isimportant in the development of type 2 diabetes, although β-cell dysfunctioncharacterized by an inability to secrete adequate amounts of insulin to overcomeinsulin resistance also contributes to the pathogenesis of type 2 diabetes Thus thedevelopment of insulin-secreting cells would provide an effective therapy fortype 1 and, possibly, type 2 diabetes

3.1.1 P ANCREAS D EVELOPMENT

The molecular mechanisms of pancreatic development provide insight intothe transcription factors needed to initiate the hierarchical cascade of geneexpression that results in differentiation along an islet cell lineage This knowl-edge will facilitate developing strategies to generate insulin-secreting cells fromstem cells The molecular and cellular mechanisms important for pancreatic

development have been the subject of several recent reviews (14,18–20) A brief

overview is presented here

Pancreatic islet development is a complex process dependent on multiplefactors, including expression of a series of transcription factors important for celldifferentiation and transmission of signals generated from surrounding mesen-chyme and blood vessels Differentiation of endoderm precursor cells into islets

is controlled by a cascade of transcriptional events directed by a series of tion factors that are expressed in a temporal and cell-specific pattern (Fig 1).Expression of Pdx-1, a homeodomain protein, is important for early pancreatic

transcrip-development, because mice and humans homozygous for mutations in the Pdx1

gene are apancreatic Subsequently, neurogenin3 (ngn3) expression is importantfor the differentiation of pancreatic endocrine cell types Null mutations of the

ngn3 gene abrogate islet development in mice (21,22) Additional transcription

factors, including NeuroD1/β 2 and Pax 6, also affect islet cell development,whereas Pax 4, Nkx2.2, and Nkx6.1 are important for β-cell development, al-though some of these factors also contribute to the differentiation of α, δ, orpancreatic polypeptide cells in islets As indicated in Fig 1, many of these tran-scription factors are expressed not only during development but also in differen-tiated adult islet cells

3.1.2 I NSULIN -S ECRETING C ELLS F ROM ES C ELLS

As described elsewhere (Chapter 8), protocols to induce the differentiation of

ES cells into insulin-secreting cells have been developed (23–25) To date, the

efficiency of generating insulin-secreting cells using these protocols has beenlow, and the cells have, in general, been relatively hypofunctional compared withnative islets One approach to enhance the differentiation process has been toexpress transcription factors important in islet development

The impact of constitutively expressing either Pdx-1 or Pax4 in ES cells was

recently described (26) Pdx-1 functions at multiple levels of pancreatic

devel-opment It is important not only for development of the exocrine and endocrinepancreas, but it is also important for maintaining the differentiated β-cell pheno-type, as it regulates the expression of several genes important for β-cell function,including the genes that encode insulin, the glucose transporter GLUT2, and

glucokinase (14,18–20) Pax4 is a paired domain homeobox transcription factor

that is important for committing endocrine precursor cells along the β- and δ-cell

lineage, because islets from mice with a null mutation of the Pax4 gene lack β and

δ cells (14,18–20) Three different approaches have been used to differentiate

native ES cells and ES cells expressing either Pdx-1 or Pax4: (1) spontaneousdifferentiation in embryoid bodies followed by adherent culture in standardmedium, (2) selection of nestin-positive cells and differentiation using a protocol

similar to that described by Lumelsky et al (24), and (3) use of nestin-positive cells in histotypic culture that promotes the generation of spheroids (26) In cells

Fig 1 Model for the role of transcription factors during islet differentiation The posed role for different transcription factors in islet differentiation is shown For simplic- ity, the association of a single transcription factor with different developmental events is based on the timing of their expression or the timing of their predominant role in differ- entiation Any given factor likely functions at multiple steps during differentiation, and expression of multiple factors is probably required at each step of differentiation Also shown are differentiated adult islet cells Below each cell is the hormonal product of that cell type and the transcription factors that are expressed in the differentiated adult δ, β,

pro-α, and pancreatic polypeptide cells.

undergoing spontaneous differentiation, Pax4- and Pdx-1-expressing cells erally showed increased expression of genes encoding transcription factors andother proteins important for or characteristic of differentiated islet cell function.Moreover, the amount of insulin mRNA and percentage of cells expressinginsulin was increased in the Pdx-1- and Pax4-expressing cells, although theimpact of Pax4 was greater than that of Pdx-1 After the selection and differen-tiation of nestin-positive cells, approximately 80% of Pax4-expressing cellsproduced insulin Growth of cells in histotypic culture resulted in spheroidscontaining cells with insulin-positive granules, albeit at a density lower than thatpresent in adult β cells When transplanted into diabetic mice, differentiatednestin-positive Pax4-expressing and wild-type ES cells were equally efficacious

gen-in restorgen-ing euglycemia Thus expression of transcription factors important forβ-cell development and differentiation augments the in vitro differentiation of

ES cells into insulin-secreting cells, although the functional consequences invivo remain unclear One problem with the approach described previously is thattranscription factor expression during development is dynamic Indeed, Pax4 isimportant for β-cell differentiation during development, but it is essentiallyabsent in adult murine β cells (27) Pdx-1 expression is relatively uniform early

in development, but is later heterogeneous with high levels in β cells and lowerlevels in undifferentiated precursor cells (19) Thus constitutive expression fails

to reproduce the dynamic regulation of transcription factor expression istic of cellular differentiation

character-3.1.3 I NSULIN -S ECRETING C ELLS F ROM T ISSUE S TEM C ELLS

An alternative approach to using ES cells is to redirect the differentiation ofadult stem cells along an islet lineage One means of accomplishing this has been

to use cells of endodermal origin This has been attempted using IEC-6 cells,which are immature rat intestinal stem cells that exhibit an undifferentiated

morphology and limited expression of intestinal-specific genes (28) Various

approaches have been used to direct the differentiation of these cells into secreting cells Stable and constitutive expression of Pdx-1 in IEC-6 cells causedthem to assume an enteroendocrine cell phenotype capable of expressing sero-

insulin-tonin, cholecystokinin, gastrin, and somatostatin (29) To direct these cells along

an islet cell lineage, the Pdx-1-expressing cells were subsequently treated with

betacellulin (30,31) Betacellulin is a member of the epidermal growth factor

family of peptides that is expressed in adult and fetal pancreas, signals throughthe ErbB family of tyrosine kinase receptors, and stimulates the proliferation ofmultiple cell types, including β cells (32,33) Several lines of evidence suggestthat betacellulin plays a key role in islet cell proliferation or differentiation.Betacellulin enhances pancreatic regeneration after a 90% pancreatectomy byincreasing β-cell proliferation and mass (34) It also increases DNA synthe-

sis in human fetal pancreatic epithelial cells and enhances β-cell development

in fetal murine pancreatic explant cultures (33,35) Treatment of

PDX-1-express-ing IEC-6 cells with betacellulin resulted in insulin expression and the formation

of secretory granules However, insulin secretion was neither glucose-dependent

nor stimulated by arginine (30,31) Among the transcription factors induced by

betacellulin treatment was Isl-1 Isl-1 is an LIM homeodomain factor that isimportant early in pancreatic development and is expressed in pancreatic epithe-

lium and mesenchyme surrounding the pancreas (36) It is also expressed later

in development in postmitotic endocrine cells and is present in mature islet cells

(36) Its role in islet function is unclear Overexpression of Isl-1 in ing cells also resulted in insulin expression (30,31) Transplantation of IEC-6 cells

Pdx-1-express-expressing both Pdx-1 and Isl-1 into diabetic rats transiently decreased the blood

glucose level, although euglycemia was not restored (30) These studies suggest

that expressing specific transcription factors in tissue stem cells can redirect theirdifferentiation along an islet lineage, but that additional factors will be needed

to fully differentiate the cells

Liver is a second endoderm-derived tissue that has been used as a source ofcells that can be directed to differentiate into islets Like pancreas, liver is derivedfrom ventral endoderm, and both tissues express members of the hepatocyte

nuclear family and exhibit glucose responsiveness (37) Indeed, it has been

sug-gested that there is an endodermal progenitor cell common to liver and pancreas

(38) In vivo expression of transcription factors has been used to differentiate liver cells into insulin-secreting cells (37) Adenoviral-mediated expression of Pdx-1 has successfully generated insulin-producing cells in liver (39,40) After

expression of Pdx-1, liver produced not only insulin, but also other islet genes,including those encoding glucagon, somatostatin, and islet amyloid polypeptide

Expression of these genes, as well as the Pdx1 gene, was prolonged as Pdx-1,

insulin, and somatostatin expression was present 6–8 months after the initialinfection Glucagon expression was extinguished after about 4 months Pro-longed expression of Pdx-1, and presumably other islet proteins, appeared to be

due to auto-induction of the native Pdx1 gene by Pdx-1 expressed from the adenoviral vector (40) After Pdx-1 expression, the insulin content of the liver

was increased 10- to 30-fold, but this was still only 1.3–3% of the insulin content

of pancreas (40) Insulin produced by the liver was functional in that it was able

to treat and prevent diabetes induced by streptozotocin, a β-cell toxin (39,40).The cells producing insulin were distinct from those that produced glucagon andwere localized in proximity to the central vein Mature hepatocytes reside in thisregion of the liver, although, because only a small percentage of infected cellsexpressed insulin, only a small subpopulation of cells appears to be capable oftransdifferentiation The nature of these cells that undergo transdifferentiation isnot clear

A similar approach has been used to develop insulin-producing cells from

epithelial progenitor cells derived from fetal liver (41) These cells express

markers of hepatocytes, bile ducts, and oval cells and are capable of

differenti-ating into mature hepatocytes in vivo (42) Oval cells are thought to represent hepatic stem cells (43) Transduction of these progenitor cells with a lentivirus

that constitutively expresses mRNA encoding Pdx-1 results in partial

differen-tiation along an islet lineage (41) Despite expression of Pdx-1, these cells

con-tinued to express hepatocyte markers, including glycogen, dipeptidyl peptidase

IV, and γ-glutamyl transpeptidase Autoinduction of the endogenous Pdx1 genewas again evident, and some transcription factors present in adult β cells (e.g.,NeuroD1, Nkx6.1) were also expressed, whereas others such as Nkx2.2 and Pax6

were absent (41) Interestingly, neurogenin3, which is present in developing but

not mature islets, was also present Finally, insulin and the prohormoneconvertases PC1/3 and PC2 as well as islet amyloid polypeptide, glucagon,pancreatic polypeptide, and elastase were expressed Thus proteins present inboth the endocrine and exocrine pancreas were produced It has not been estab-lished whether these different hormones and enzymes are coexpressed by thesame or different cells Importantly, these cells exhibit glucose-stimulated insu-lin secretion, albeit with a curve that is shifted to the right compared with nativeislets This may reflect a lack of expression of GLUT2 and glucokinase andexpression of only the Kir6.2 subunit of the ATP-sensitive potassium channelthat is important for insulin secretion Importantly, these cells appeared to secretemature processed insulin and were able to reverse streptozotocin-induced diabetes

In studies using an adenoviral vector capable of higher and more prolongedexpression, in vivo Pdx-1 expression in the liver had a different effect In thiscircumstance, insulin-producing cells were present, but cells exhibiting charac-teristics of exocrine cells, including expression of trypsin, were also present

(44,45) Interestingly, insulin and trypsin were coexpressed by the same cells, and the latter induced a severe hepatitis (44,45) In contrast, use of this same

adenoviral vector to express the transcription factor NeuroD1/Beta2 andbetacellulin resulted in the formation of islet clusters capable of reversing

streptozotocin-induced diabetes (44,45) The islet-like clusters were, in general,

localized immediately underneath the liver capsule Thus the cells from whichislet-like structures were generated appeared to be distinct from those in theproximity of the central vein that differentiated into insulin-secreting cells fol-lowing Pdx-1 expression After expression of NeuroD1 and betacellulin, gluca-gon, somatostatin, and pancreatic polypeptide were also present in the islet-likestructures Unlike native islets, individual cells in the islet-like structures pro-duced multiple hormones Other genes characteristic of mature islets were alsoexpressed, including those encoding the prohormone convertases PC1/3 and

PC2 and the Kir6.2 and SUR1 subunits of the ATP-sensitive potassium channel

(44,45) Insulin granules were also present in the cells.

3.2 Steroidogenic Cells

Another endocrine gland susceptible to destruction by autoimmunity, tion, and bleeding is the adrenal gland Because oral replacement of cortisol doesnot accurately reproduce the pattern of cortisol secretion by the native adrenalgland, the generation of adrenal cells from stem cells would be of therapeuticbenefit

infec-The only transcription factor that has been expressed in ES cells to help directdifferentiation along a steroidogenic cell lineage is steroidogenic factor 1 (SF-1)

(46) SF-1 is an orphan member of the steroid receptor superfamily (reviewed in (47) It is expressed in a variety of tissues, including the adrenal cortex, testis

(Sertoli cells), ovary (granulosa and theca cells), the placenta, and the pituitaryand hypothalamus During development, SF-1 is expressed in the urogenitalridge as early as embryonic day 9 in mice, and its role in the differentiation ofsteroidogenic tissues is demonstrated by the absence of adrenal glands and

gonads in mice with a null mutation of the SF-1 gene (48,49) In humans, mutations in SF-1 are associated with hypogonadism and hypoadrenalism (47).

Among the targets of SF-1 are the genes that encode the steroidogenic

cyto-chrome P450 enzymes (47).

Given the role of SF-1, it is not surprising that its expression in ES cells directs

their differentiation toward a steroidogenic phenotype (46) The morphology of

ES cells stably transfected with a vector expressing SF-1 changes from fringent spheres into flat, phase-dull sheets despite the continued presence ofmouse embryo fibroblast feeder cells and leukemia inhibitory factor, both ofwhich prevent ES cell differentiation Among the factors known to induce ste-roidogenesis in steroidogenic cell lines are retinoic acid and cyclic adenosine 5′-monophosphate, which is the downstream effector of hormones such asadrenocorticotropic and luteinizing hormones Treatment of the SF-1–express-ing ES cells with a cyclic adenosine 5′-monophosphate analogue with or withoutretinoic acid markedly increased expression of the rate-limiting steroidogenicenzyme P450 side-chain cleavage (P450scc), an effect not observed in native ES

bire-cells (46) Moreover, in bire-cells provided with 20α-hydroxycholesterol, a substrate

for P450scc, progesterone was synthesized in amounts proportional to the sion of P450scc mRNA It is important to note that this change in cell phenotypeoccurred despite the continued presence of mouse embryo fibroblasts and leuke-mia inhibitory factor Thus SF-1 expression is capable of initiating a programthat converts ES cells into steroidogenic cells and may serve to augment thedevelopment of steroidogenic tissues from stem cells

expres-4 CONCLUSION

The studies described here indicate that transcription factor expression has thepotential to direct or augment stem cell differentiation As demonstrated, expres-sion of a specific transcription factor can initiate a genetic program typicallyactivated by inductive factors elaborated in vivo by surrounding tissues and cells,thus allowing differentiation to proceed in vitro One of the problems with thisapproach, however, is that the constitutive expression of transcription factors isnot able to reproduce the dynamic expression of transcription factors that ischaracteristic of the differentiation process This may interfere with the finalmaturation of cells or alter cell function Approaches that have been used toaddress this concern are using vectors (e.g., adenoviral vectors) in which expres-sion is time-limited or vectors that allow inducible expression of the gene ofinterest Clearly, expressing transcription factors in differentiating stem or pro-genitor cells will provide important insight into the genetic programs responsiblefor differentiation along specific cell lineages and has the potential to facilitateongoing efforts to develop means to differentiate stem cells into specific hor-mone-secreting cells that will be available for cell replacement therapy

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