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The Production and Directed Differentiation of Human Embryonic Stem Cells

Monash Immunology and Stem Cell Laboratories, Monash University, and Australian Stem Cell Centre, Clayton, Victoria 3800, Australia

Correspondence: Address all correspondence and requests for reprints to: Alan Trounson, Ph.D., Director, Monash Immunology and Stem Cell Laboratories, STRIP Building, Monash University, and Australian Stem Cell Centre, Wellington Road, Clayton, Victoria 3800, Australia. E-mail: alan.trounson{at}med.monash.edu.au

	Abstract

Top Abstract I. Introduction II. Deriving and Maintaining... III. Differentiation of hESCs IV. Patient-Specific Stem Cells V. Conclusions References

 
Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of early progenitors in this lineage. There are reports of enriched cultures of keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurons, hepatic progenitors, and cells that have some markers of gut tissue and pancreatic islet-like cells. The prospects for use of hESC derivatives in regenerative medicine are significant, and there is much optimism for their potential contributions to human regenerative medicine.

I. Introduction

II. Deriving and Maintaining Human Embryonic Stem Cells (hESCs)

A. Selection of embryos for deriving hESCs

B. Derivation of hESCs

C. Genetic manipulation of hESCs

D. Markers of hESCs

III. Differentiation of hESCs

A. Directing differentiation (Table 1)

IV. Patient-Specific Stem Cells

V. Conclusions

	I. Introduction

Top Abstract I. Introduction II. Deriving and Maintaining... III. Differentiation of hESCs IV. Patient-Specific Stem Cells V. Conclusions References

Embryonic germ cells may be formed from the primitive gonadal ridges of the developing embryo or fetus (6–9 wk gestation in the human) and have many of the pluripotential properties of ESCs (2). ASCs do not have the range of differentiation properties that ESCs are capable of, and most are uni- or multipotential, usually forming only the cell types of the lineage of their origins. Some ASCs such as bone marrow stromal cells or mesenchymal stem cells (MSCs) will exhibit plasticity for colonizing a variety of tissues under some experimental situations and in response to tissue damage and inflammation (3). A rare type of bone-marrow-derived cell that demonstrates multiplicity of lineage differentiation may be selected in the laboratory, and this cell has been termed "multipotential adult progenitor cell" (4). A similar type of multipotential stem cell can be found in human cord blood (5) and amniotic fluid (6). All these cells have an immuno-phenotype that is representative of a subtype of MSCs, and further classification of markers may identify a family of MSCs that have plasticity to form neural and muscle cell types as well as the recognized MSC lineages of adipose, cartilage, and bone tissues.

	II. Deriving and Maintaining Human Embryonic Stem Cells (hESCs)

Top Abstract I. Introduction II. Deriving and Maintaining... III. Differentiation of hESCs IV. Patient-Specific Stem Cells V. Conclusions References

 
A. Selection of embryos for deriving hESCs
The clear preference for deriving hESCs would be from euploid embryos selected after embryo biopsy for preimplantation genetic diagnosis. This technique involves chromosomal identification by multicolor fluorescent in situ hybridization (FISH). Usually one or two cells are removed from eight-cell human embryos before compaction (60–72 h after insemination) and five to 14 chromosome numbers examined by multicolor fluorescent DNA tags (Fig. 1). Usually biopsied embryos diagnosed as monosomic or trisomic on the basis of fluorescent in situ hybridization are discarded. However, some embryos are mosaic for chromosomal numbers or are chromosomally chaotic. Very few of these embryos can develop normally in vitro or in vivo (7). However, hESCs that were trisomic for chromosome 13 or were triploid have been derived from human embryos, which indicates that some of the aneuploidies observed in human embryos are compatible for ESC development (8).

View larger version (24K): [in this window] [in a new window]   FIG. 1. IVF embryos and ESCs. ICSI, Intracytoplasmic sperm injection.

B. Derivation of hESCs
Human ESCs are formed from mechanically or immuno-surgically isolated ICM of preimplantation-stage blastocysts produced for the treatment of infertile couples (20, 21) (Fig. 1) but may also be derived from earlier morula-stage human embryos (22), or intact blastocysts, after the removal of the glycoprotein shell known as the zona pellucida in an acidified solution or by enzymatic digestion in pronase (23). There appears to be very little difference in the efficiency of producing hESCs from these different stages of embryo development when grown on embryonic fibroblast feeder cells. Mouse ESCs (mESCs) may be maintained in the presence of leukemia inhibiting factor (LIF) in culture in vitro without feeder cell support, but this is not the case for hESCs (20, 24). It is also possible to direct mESCs into a trophectoderm lineage and to establish permanent trophectoderm cell lines (25, 26), but this has not been easily replicated for hESC differentiation despite the apparent expression of some trophectodermal markers in response to culture with bone morphogenic protein 4 (BMP4) (27).

Colonies of hESCs differ from the ICM in a number of ways. Firstly, ICM cells retain a memory for axes, dorsal-ventral, anterior-posterior, and left-right axes, that enables the differentiating cells to have position relationships that guide the differentiation, expansion, and integration of cell types required to form an organism. It is generally considered that ESCs are an epiblast derivative, or even a type of germ stem cell (28), that can be maintained as an immortal and pluripotential cell type under strict laboratory conditions, in the presence of secretory products of embryonic, or adult, somatic cells. Importantly, self-renewal of hESCs appears to involve the Wnt family signaling pathway (29) and probably other pathways that involve basic fibroblast growth factor (bFGF) and TGF-ß.

In 1998, Thomson et al. (10) were the first to report the successful derivation of hESCs from preimplantation human embryos. Their report followed extensive studies by Thomson and colleagues (30, 31) on the production of rhesus and marmoset ESCs. Intact blastocysts and mechanically isolated ICMs grown on mouse embryonic fibroblasts (STO cells) were studied by the research group in Singapore from 1994–1996, and these cultures resulted in cell lines that differentiated after several passages in vitro (21). The methods finally used successfully to establish hESC lines were described by Reubinoff et al. (11). These methods were similar to those described by Thomson et al. (10) and involved the isolation of ICM clusters from human blastocysts by immunosurgery and their coculture with mitotically inactivated murine embryonic fibroblasts (MEFs). The hESCs form typical colonies of undifferentiated cells that need to be passaged weekly or, more often, as mechanically dissected colonies of 10 cells or more. Additional hESC lines (32) have been derived by similar methods. More recently hESCs have also been derived under feeder-free conditions using cell-free lysates of MEFs (33).

The selection criteria used for choosing human embryos for deriving hESCs will determine the eventual success rates for their production. Small numbers of blastocyst-stage embryos grown in coculture with human oviductal epithelial cells were used by Reubinoff et al. (11) to produce six hESC lines after preliminary experiments involving around 30 embryos (20). The six hESC lines were derived from 12 blastocysts. This very high success rate of producing hESCs can be compared with the use of much larger numbers of embryos (blastocysts) by others (34). It is probable that about 50% of human embryos have chromosomal abnormalities (35), and it would be expected that these genetic errors would limit the success rate of hESC production. It is also difficult to establish hESCs from monosomic or trisomic embryos, with less than 10% made from human embryos diagnosed as aneuploid (36). Interestingly, two hESC lines produced from trisomic embryos reverted to diploidy, indicating the embryos were probably mosaic (36). A large number of hESC lines have been produced from excess human IVF embryos by some IVF clinics (Fig. 1); for example, Kukharenko et al. (37) reported 46 new hESC lines made from morulae, blastocysts, and ICMs isolated from blastocysts (38). There was apparently little difference between stages of preimplantation human embryos in their capacity to form hESC lines (22). A more recent comparison of mechanical isolation of ICMs and plating whole blastocysts for deriving new hESC lines showed that mechanical isolation is more efficient (39). The use of antiserum raised in animals for immunosurgery to isolate ICMs is undesirable.

Mosaic human blastocysts have been constructed by aggregating uninuclear cells of poor-quality embryos that would be normally discarded by IVF clinics because they lack full developmental potential (40). However, the variation in genetic composition of these mosaic embryos would limit their usefulness as hESCs. It has also been shown that single blastomeres of eight-cell preimplantation mouse embryos can be used to derive ESCs (41), without necessarily compromising the developmental potential of the early cleavage-stage embryo (Fig. 1). This is surprising because ICM cells are formed from internalized cells within the morula-stage embryo, and compaction of the two to four cells formed from such biopsies wouldn’t enable a cell to be internalized and depolarized as required for producing ICM cells (42). Growth of biopsied cells from early cleavage-stage mouse embryos has been shown in the past (43), but the single-cell outgrowths were not cultured with appropriate feeder cells for producing ESCs.

The long-term stability of hESCs is an important issue, and although normal karyotypes can be maintained for extended culture times in vitro (44, 45), others have reported instability of chromosomes 12 and 17 in conditions that are known to stress hESCs (46, 47). It is important to reassess karyotypes regularly for hESCs, particularly those passaged by enzymatic digestion into single-cell suspensions because they may continue to express pluripotent markers even when they have become aneuploid. Similarly, there may be a need to monitor for mutations that may influence differentiation and tumor formation in vivo and might appear in critical genes such as the oncogene family. It is apparent that there is considerable stability in the epigenetic state of some imprinted genes of hESCs during long-term culture in vitro (48).

There is a wide range of feeder cells that are appropriate for the maintenance of hESCs, including murine fetal fibroblasts (e.g., STO cells) (10, 11) and human cell lines, including human embryonic fibroblasts (12), human uterine endometrium (49), human foreskin fibroblasts (13), human adult bone marrow cells (50), and differentiated hESCs (51, 52). Other cell lines, including commercially available human cells, are also in use for the maintenance of hESC and may also be appropriate for deriving the hESC from human embryos. There hasn’t as yet been an analysis of transcription profiles of hESCs derived on varying feeder cells or reports of common active factors that maintain hESCs that are associated with these feeder cell types. Conditioned culture medium from MEFs can maintain hESCs when grown on Matrigel or laminin extracellular matrices (29, 53, 54).

Serum-free culture systems containing serum substitutes and FGF-2 may be used for propagation of hESCs and have reduced spontaneous differentiation. Amit et al. (55) showed that hESCs can be maintained in medium containing serum replacement, FGF-2, TGF-ß, LIF, and fibronectin extracellular matrix. Recently Pebay et al. (56) have demonstrated that sphingosine-1-phosphate and platelet-derived growth factor are active serum components that can replace the need to use serum for hESC culture. These observations show that signaling pathways for hESC renewal may be activated by tyrosine kinases synergistically with those downstream from lysophospholipid receptors.

Serum-free and feeder layer-free conditions have been reported for the long-term maintenance of hESCs. Amit et al. (57) reported on hESCs grown in medium containing 15% serum replacement and growth factors, TGFß1, LIF, bFGF, and fibronectin. After 47–50 passages, the hESCs maintained euploidy and pluripotentiality. A hESC line has also been established on plastic dishes coated with extracellular matrix, which was dried and sterilized, from mouse embryonic fibroblasts (33). The medium composition was similar to that reported by Amit et al. (57). The presence of bFGF is considered essential in these feeder-free culture systems (58). Human serum has also been used to coat tissue culture dishes, then dried and used for culture of hESCs in the presence of medium conditioned by hESC-derived mesenchymal cell types (59). The medium used for conditioning was again similar to that reported by Amit et al. (57).

Optimizing culture conditions for hESCs is extremely important and is discussed in considerable detail by Hoffman and Carpenter (34). It is likely that there will be a change to the production of hESCs under the rigid regulations involved for Good Manufacturing Practice, and this will possibly include the need for screening of patients for the prevention of transmission of infections, Good Manufacturing Practice production of embryos from IVF clinics, and completely humanized culture and preparation methods that do not involve any animal reagents. All methods will be exactly defined and subject to quality control procedures and will have a minimal human handling component and consequently will involve increased use of robotics technology and bioprocessing. Bulk culture systems for hESCs are still in their infancy, and much work is needed to improve these systems for optimal production of hESCs and their derivatives.

C. Genetic manipulation of hESCs
Clonal derivation of hESCs is difficult, and the efficiency is extremely low (55). However, it is possible to transfect hESCs with DNA constructs, and this is important for determining the role of transcription factors for the renewal and differentiation of hESCs. Identification of specific gene expression by reporter genes enables purification of cells of interest in differentiating cultures and the tracking of hESC derivatives in mixed cell cultures or when transplanted into animal models. Conventional transfection methods have been successful (60), as have lentiviral methods (61, 62). Integration of reporter genes into controlling elements of specific genes or the approach of gene knock out or knock in used for functional genomics is very difficult because of the inability to clone hESCs. However, Zwaka and Thomson (63) have shown that it is possible to electroporate hESCs to achieve homologous recombination of hESC colony fragments. Gene function may be more appropriately determined in hESCs by using small inhibitory RNAs (64) to control renewal, differentiation, apoptosis and other mechanisms involved in cell function and response to internal and external stimuli.

D. Markers of hESCs
Sperger et al. (65) have reported that, by microarray analysis, 330 genes are highly expressed in common in hESCs and human embryonal carcinoma cells and seminomas. This included POU5F1 (Oct4) and FLJ10713, a homolog highly expressed in mESCs (66). Among those genes only highly expressed in hESCs and human embryonal carcinoma cells included a DNA methylase (DNMT3B), which functions in early embryogenesis (67), and Foxd3, a forkhead family transcription factor that interacts with Oct4, which is essential for the maintenance of mouse primitive ectoderm (68). Sox2 is also highly expressed and is known to be important in pluripotentiality (69). Serial analysis of gene expression (SAGE) has been reported by Richards et al. (70) and has been compared with some cancer SAGE libraries. As expected, Oct4, Nanog, and Sox2 transcripts appear abundantly, but there were differences between hESCs in some other transcript abundance (e.g., Rex-1). Expression profiles were also similar to human embryonal carcinoma cell lines in this study.

Markers that are now recognized as important for hESC pluripotentiality include Oct4, Nanog, Sox2, Foxd3, Rex1, and UTF1 transcription factors; TERF1, CHK2, DNMT3 DNA modifiers; GFA1 surface marker; GDF3 growth factor; TDGF1 receptor; and Stella and FLJ10713 (71).

For characterization of hESCs, it is common to report one or more of the following: Oct4 expression, alkaline phosphatase and telomerase activities; stage-specific embryonic antigens 3 and 4; hESC antigens TRA-1–60, TRA-1–81, GCTM-2, TG-30, and TG-343; and CD9, Thy1, and major histocompatibility complex class 1 (MHC-1) (72). Other stem cell antigens are also sometimes reported, e.g., AC133, c-kit (CD117), and flt3 (CD135), but these are frequently only expressed in a proportion of the hESC population, making them potential derivatives of interest in the heterogeneous hESC cell population [see the discussion in the review by Hoffman and Carpenter (34)]. The presence of Oct4 expression alone may be misleading, as this transcription factor takes some time to shut down RNA transcription in differentiating hESCs and is also found in other pluripotent cell populations (e.g., embryonic germ stem cells), as well as some adult and fetal multipotential stem cells. Target genes of Oct4 include Rex-1, Lefty-1, PDGFalfaR and Utf-1, and those cooperating with Oct4 include Sox2 (34).

	III. Differentiation of hESCs

Top Abstract I. Introduction II. Deriving and Maintaining... III. Differentiation of hESCs IV. Patient-Specific Stem Cells V. Conclusions References

 
Faster spontaneous differentiation of hESC colonies occurs in vitro in prolonged cultures and in the absence of actively secreting feeder cells. Early differentiation events may be observed in many hESC colonies within a week of passage (17), and heterogeneity in markers of stem cell pluripotentiality (e.g., Oct4 expression) can be observed in early differentiating hESC colonies. When hESCs are permitted to overgrow in two-dimensional culture, cells begin to pile up and differentiation begins at the borders of the colony and also in the central piled-up areas of the multiplying colony (17). A wide range of differentiating cell types can be observed in these flat cultures, including ectodermal neuroectoderm, mesodermal muscle, and endodermal organ tissue types (11) (Fig. 2).

View larger version (26K): [in this window] [in a new window]   FIG. 2. hESCs and their use in research.

In the absence of tests for forming tissue chimeras by recombining ESCs with embryonic cells as used in the mouse, the classical assay for pluripotentiality of hESCs is teratoma formation in xenografts to immune-compromised mice. Attempts to derive human or human-animal chimeras would be unethical in the human. When hESCs are transplanted into animal tissues, they rapidly form solid teratomas of advanced development and of mixed tissue lineage. The tissues that are recognized are often primitive but well-organized examples of embryonic or fetal organs. Normally hESCs are transplanted under the kidney or testis capsule and recovered within 1–8 wk for histological examination (10, 11). The teratomas formed are interesting because they are mainly of human origin but also contain mouse microstructures with histotypic appearance of the differentiating human tissue (80). This suggests that there is communication and instruction taking place in both directions, i.e., mouse tissue accelerates hESC differentiation and hESC derivatives of the teratoma instruct mouse tissue at the site of transplantation. Hence, the microenvironment will influence the direction and the rate of differentiation of ESCs, which is something that needs to be recognized in construction of differentiation and transplantation systems for stem cells.

A. Directing differentiation (Table 1)
The enhancement of differentiation toward a specific lineage (71, 81) can be achieved by the following: activating endogenous transcription factors; transfection of ESCs with ubiquitously expressing transcription factors; exposure of ESCs to selected growth factors; or coculture of ESCs with cell types capable of lineage induction. ESCs may be induced to form the lineage of interest by a combination of growth factors and/or their antagonists (82). These instructors of lineage formation accelerate differentiation in vitro and mimic the markers of natural developmental pathways.

Formation of ectodermal derivatives is very common in spontaneously differentiating hESCs (11, 77) and is commonly considered a developmental default pathway. The neural differentiating pathway can be enhanced in cultures (83) and the neural progenitors transplanted into the ventricles of the brain of newborn mice, resulting in diffuse migration of human neurons and astrocytes into the brain parenchyma and the presence of human neural stem cells passing along the olfactory rostral migratory pathway (77, 84). These hESC-derived neurons respond to neurotransmitters, generate action potentials, and make functional synapses (83).

Oligodendrocytes may also be produced in enriched hESC culture derivatives using bFGF and epidermal growth factor, followed by the additional supplementation of all-trans-retinoic acid (RA). The oligodendrocyte precursors produced are able to mature and remyelinate neurons of the shiver mouse model (85). Dopaminergic neurons can be formed from hESCs (86, 87), and these are of interest in preclinical transplantation experiments. Motor neurons can also be produced using the multistep method used for this differentiation pathway in the mouse (88) that utilizes RA and FGF2, then RA and sonic hedgehog (SHH), and finally brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor, IGF-I, and low levels of SHH. There is optimism that transplantation of these hESC-derived neurons and glia may be able to repair injured spinal cord in cases of severe trauma (89).

The directed differentiation of hESCs into neuroectoderm may be efficiently achieved using Noggin, which is an antagonist to BMP signaling that is involved in the paracrine loop that drives hESCs into flattened epithelial that express genes characteristic of extra-embryonic endoderm. These cells are a human yolk sac cell type that proliferates in spontaneously differentiating cultures under the influence of BMP2 produced by hESCs. The "Noggin cultures" are capable of renewal in culture as relatively homogeneous colonies of neuroectoderm and show facile conversion to neurons or glia in the appropriate culture systems (90). On the other hand, prolonged culture of hESCs in serum-free medium with BMP4 will induce flat epithelial cells that express genes (e.g., MSX2) and proteins (e.g., human chorionic gonadotropin) associated with trophoblast or placental development. These cells later show syncytial giant cell morphology typical of placental derivatives (27).

Coculture of nonhuman primate ESCs with the mouse bone marrow mesenchymal PA6 cell line that produces stromal cell-derived inducing activity (SDIA) will produce midbrain neuronal cells that are tyrosine hydrolase positive (TH⁺) and express nurr1 and LMX1b genes (91, 92). In these differentiating cultures, pigmented retinal epithelium could also be recognized. Manipulation of culture conditions with BMP4 induces epidermogenesis or neural crest cells and dorsal-most central nervous system cells. Suppression of SHH promotes motor neuron formation (93). Perrier et al. (87) showed that hESCs can also be directed into midbrain dopamine neurons when grown with mouse bone marrow mesenchyme (MS5 and S2 cell lines). They showed the sequential expression of the key transcription factors Pax2, Pax5, and engrailed-1 in response to a series of growth factors and patterning molecules (FGF8, SHH, ascorbic acid, and BDNF). High yields of TH⁺ neurons were obtained in their studies. Interestingly, SDIA does not promote differentiation of all types of neural stem cells into dopaminergic neurons in the same way as for hESCs (94). Differences in hESC lines in response to coculture with PA6 cells was noted by Park et al. (95). Even in hESC lines that did produce TH⁺ dopaminergic neurons, few survived when transplanted to hemi-Parkinsonian-grafted animals (95), an observation supporting previous reports (96).

TH⁺ neurons have also been derived by selection of neuroectodermal rosettes in differentiating cultures of hESCs (97, 98). Neural precursors and dopaminergic neurons were formed by initial culture in medium conditioned by HepG2 liver tumor cells, followed by conventional serum-free culture in medium containing FGF2. TH⁺ human neurons survived up to 8 wk after transplantation in 6-hydroxydopamine-lesioned rats (98). Neuroectoderm selected from spontaneously differentiating hESCs that were grown into neurospheres in serum-free medium differentiated into dopaminergic neurons in vivo when grafted into the striatum of Parkinsonian rats but at low efficiency (99). Transplanted neurons were identified 12 wk after transplantation, although they were not proliferating, and only partial correction of drug-induced rotational behavior was observed. These data are encouraging but clearly a long way from clinical studies in human Parkinson’s disease patients.

Yan et al. (100) have shown that exposure of the FGF2-expanded neuroepithelial cells of hESC derivation to FGF8 and SHH promotes differentiation of dopaminergic neurons with a forebrain phenotype, but early exposure to FGF8 during neuroepithelial specification promotes the midbrain phenotype and subsequent midbrain dopaminergic neurons. Hence, the sequence of instruction by FGF8 and SHH determines the neuronal subtype that is of some significance for cell therapies and drug screening studies. The type of neuronal progenitor produced by recapitulation of patterning pathways using natural sequences of developmental inducers will be critical for strategies to repopulate specific degenerative lesions such as those observed in Parkinson’s disease.

Coculture methodologies have also been used to produce differentiated cardiomyocytes from hESCs. Mummery and colleagues (101, 102) showed that 15–20% of cultures of hESCs grown with the mouse visceral endoderm cell type (END-2) will form beating heart muscle colonies, and this has been substantially increased in more recent experiments. Beating heart muscle cells derived from hESCs express cardiomyocyte markers including -myosin heavy chain, cardiac troponins, and atrial natriuretic factor as well as transcription factors typical of cardiomyocytes, e.g., Nkx2.5, GATA4, and MEF3 (78, 79, 101). These cells respond to pharmacological drugs, and the action potentials of cardiomyocytes produced in this system most commonly resemble that for human fetal left ventricular cardiomyocytes but are distinctly different from those of mouse cardiomyocytes (102, 103). Atrial- and pacemaker-like cells may also be formed in the differentiating hESC cultures. The hESC-derived cardiomyocytes are capable of integrating apparently normally when transplanted into rodent and porcine heart muscle, forming gap junction connections between hESC myocytes and the recipient mouse adult cardiomyocytes (104, 105, 106). This research has advanced the prospect of hESCs being used in the clinical treatment of cardiac infarcts.

Studies on the successful induction of mouse alveolar phenotypes from mESCs that have the morphological appearance of type II pneumocytes and that express surfactant protein C (respiratory-specific marker) by coculture with mouse embryonic fore-gut mesenchyme have also been shown to be effective for differentiation of hESCs into the respiratory lineage (107). The hESC-derived cells also expressed human surfactant protein C. This finding is encouraging for the further research on the use of hESCs for alveolar lung engraftment. Mouse ESCs can also be induced to form airway epithelial tissue when differentiated as embryoid bodies or grown on type 1 collagen, and then the resulting Clara cells grown in an air-fluid interface form a pseudostratified surface epithelium (108). These observations are of interest to repair of cystic fibrosis pathology in the upper airways of the lung.

Keratinocytes can be derived from hESCs by replating embryoid bodies (109). Cells expressing the transcription factor p63 in the periphery of the secondary cultures identify the keratinocyte progenitors that produce more mature cell types in which cytokeratin 14 and basonuclin are detected. These cells can form terminally differentiated stratifying epithelium but were not the same as keratinocyte epithelium isolated from neonatal or adult skin.

The hematopoietic lineage can be induced to form from differentiating hESCs (110). Initiating spontaneous differentiation by forming embryoid body cultures and by using a cocktail of hematopoietic cytokines and BMP-4, Chadwick et al. (111) have induced the formation of hematopoietic progenitors that could produce both erythroid and myeloid derivatives. The progenitors were immunologically similar to hematopoietic progenitors of the dorsal aorta. The growth factors used were stem cell factor, IL-3 and IL-6, granulocyte colony-stimulating factor, and Flt-3 ligand. A further enhancement of erythroid colonies can be obtained with the addition of vascular endothelial growth factor-A (112, 113). Ng et al. (114) have developed a novel hESC aggregation system that permits the sequential expression of primitive streak (MIXL1 and Brachyury) and mesoderm markers (Flk1/KDR). Approximately one in 500 hESCs will produce hematopoietic precursors using this system.

Selection of differentiating cells of the endodermal lineage has been difficult, probably because of the lack of markers of early endoderm progenitors. However, definitive endoderm may be induced in mESCs by restricting culture in serum or the exposure to activin A (115). There is much interest in the production of pancreatic ß-islet cells in the endoderm lineage because of the potential to treat diabetes. Some cells of human embryoid bodies will stain positive to insulin antibodies (116), but although they weakly express insulin-2, they do not express insulin-1 and do not stain for C-peptide and insulin-positive cells are likely to be a result of uptake up of insulin from the culture medium (117). Some insulin-producing ß-like cells can be found in spontaneously differentiating overgrowth conditions of hESCs on MEFs (118).

Insulin-producing cells can be formed from differentiating neuroectoderm in the mouse (119). Using a modified method of Lumelsky et al. (119), Segev et al. (120) have produced islet-like clusters from spontaneously differentiating hESCs. Embryoid bodies were grown for 7 d followed by plating for another week in insulin-transferrin-selenium-fibronectin medium. Disaggregated cultures were allowed to form clusters in medium containing bFGF and then exposed to nicotinamide with low glucose in suspension culture. A high percentage of insulin- and glucagon- or somatostatin-coexpressing cells were observed in the cell clusters formed, which were considered to be similar to immature pancreatic cells. Responsiveness to glucose and antagonists was lower than expected and may be due to the immaturity of the pancreatic-like cell clusters produced, similar to the poor responsiveness of fetal pancreatic ß-islet cells. Although there is clearly more work needed in the development of the pancreatic lineage and ß-cell function, progress is encouraging although incomplete.

Rambhatla et al. (121) reported differentiation of hESCs into cells expressing markers of hepatocytes (albumin, -1-antitrypsin, cytokeratin 8 and 18) that accumulate glycogen by treatment of differentiating embryoid bodies with sodium butyrate or adherent hESC cultures with dimethyl sulfoxide followed by sodium butyrate. Others have reported hepatic-like endodermal cells in embryoid bodies (122). The selection of cells with particular morphology in adherent hESC cultures differentiating in vitro may also favor endodermal populations that express markers of fetal liver (123). These data suggest that with the appropriate markers, it will be possible to select cells capable of forming liver, gut, and other endodermal tissues.

	IV. Patient-Specific Stem Cells

Top Abstract I. Introduction II. Deriving and Maintaining... III. Differentiation of hESCs IV. Patient-Specific Stem Cells V. Conclusions References

 
There is much interest in the production of patient-specific stem cells (Fig. 2) using nuclear transfer techniques to introduce somatic cell nuclei into enucleated oocytes (124). The reason for making hESCs for individual patients is for the possible establishment of immune-compatible cell derivatives for transplantation.

It is important that new disease-specific stem cells be derived from patients with cancers; neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, motor neuron disease, and multiple sclerosis; and others of unknown cause or multigenic origins. The ability to reestablish pristine hESCs that can be differentiated in the laboratory to cells that will express the disease phenotype could be a very valuable resource for screening for molecules that interfere with the disease phenotype and identifying candidate drugs or molecular pathways that may enable a whole new approach to pharmaceuticals for these patients (Fig. 2). This approach has already proven productive using mESCs (125).

It is difficult to comprehend the system that will require a patient-specific ESC for every patient and how this would be organized in an efficient and timely manner. Immune response to hESCs is muted, and they fail to stimulate proliferation of alloreactive primary T lymphocytes when transplanted into immune-competent mice (126). It is known that hESCs express only low levels of MHC-1, but this is increased 2- to 4-fold when they differentiate and are higher again in teratomas (127). Even in differentiated cells derived from hESCs, MHC-1 expression is less than in somatic cells and MHC-II is absent. Grafts do not survive when transplanted across major histocompatibility barriers, although there may be tolerance of some mismatching as in umbilical cord blood. A number of strategies have been proposed to prevent rejection of transplanted hESC-derived cell types (128). It is likely that hESCs could be used to induce immune tolerance to their own histocompatibility type by establishing bone marrow and/or thymic chimerism before therapeutic transfer of the stem cells (129). This would avoid the need to establish vast banks of hESCs for compatible cell therapies and the need to make patient-specific ESCs by nuclear transfer. There may well be other approaches for inducing pluripotentiality using cell fusion techniques that would avoid the temporary production of nuclear transfer embryos (130, 131). The production of somatic-ESC hybrids (132), which express many genes typical of pluripotential cells, results in tetraploid cells that may be unstable and have little clinical usefulness. Hence, attempts to remove the ESC nucleus before nuclear hybridization may be a more attractive approach (133).

	V. Conclusions

Top Abstract I. Introduction II. Deriving and Maintaining... III. Differentiation of hESCs IV. Patient-Specific Stem Cells V. Conclusions References

 
Human ESCs can be made from embryos that are produced in IVF clinics that are excess to the patients’ needs. They may have known disease-specific mutations that are useful for interrogating the expressed phenotype and studying how these disease states may abrogated. It is also possible to produce disease-specific stem cells by nuclear transfer from patients with cancers, neurodegenerative disease, and other disorders of unknown or multigenic and epigenetic origins for studying the influence of small molecule screens on phenotypic expression in the laboratory. These are powerful new tools for medical research in regenerative medicine. It is also now possible to direct the differentiation of hESCs into lineages of therapeutic interest and to enrich cultures for cell types of interest in transplantation and drug screening. Research at present is focused on stem cell biology and preclinical evaluation of efficiency and safety for correction of disease and injury in animal models. There is considerable optimism that hESCs will make a very major contribution to human medicine as cell therapy, vehicles for gene therapy, and drug discovery.

	Footnotes

 
First Published Online January 24, 2006

Abbreviations: ASC, Adult stem cell; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; BMP, bone morphogenic protein; ESC, embryonic stem cell; hESC, human ESC; ICM, inner cell mass; IVF, in vitro fertilization; LIF, leukemia inhibiting factor; mESC, mouse ESC; MEF, murine embryonic fibroblast; MHC, major histocompatibility complex; MSC, mesenchymal stem cell; RA, all-trans-retinoic acid; SDIA, stromal cell-derived inducing activity; SHH, sonic hedgehog; TH+, tyrosine hydrolase positive.

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