[31] Factors Controlling Human Embryonic Stem Cell Differentiation By MAYA SCHULDINER and NISSIM BENVENISTY Introduction Human embryonic stem (ES) cells are pluripotent cell lines derived from the inner cell mass (ICM) of blastocyst stage human embryos. 1,2 These cells possess self-renewal capabilities, which can be preserved through tight regulation of their growth conditions. Such regulation enables these cells to proliferate indefinitely in culture. However, once the cells are allowed to differentiate, they spontaneously develop into various cell types. 3 The differentiation process can be influenced, to some extent, by use of external 1 J. A. Thomson, J. Itskovitz-Eldor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V. S. Marshall, and J. M. Jones, Science 282, 1145 (1998). 2 B. E. Reubinoff, M. F. Pera, C. Y. Fong, A. Trounson, and A. Bongso, Nat. Biotechnol. 18, 399 (2000). 3 J. Itskovitz-Eldor, M. Schuldiner, D. Karsenti, A. Eden, O. Yanuka, M. Amit, H. Soreq, and N. Benvenisty, Mol. Med. 6, 88 (2000). Copyright ß 2003, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 365 0076-6879/2003 $35.00
Transcript
Once differentiation has been induced, RT-PCR or immunocytochem-ical techniques may be used to monitor changes in gene expression and cellphenotype. It is also very important ultimately to obtain evidence ofdifferentiated cell function. A discussion of the scope of functionaltests available for differentiated cell types is beyond the scope of thischapter, but might include tests in vitro (electrophysiology for neurons,glucose-dependent insulin secretion for beta islet cells of the pancreas) aswell as transplantation studies in vivo into developing tissue or into damagedtissue, with assessment of appropriate integration of cells into the tissue andrepair of tissue function following lesion or injury.
Acknowledgment
Work in our laboratory is supported by ES Cell International Pte., the National Health and
Medical Research Council, and the Juvenile Diabetes Research Foundation. We thank all the
members of our laboratory for their input into this chapter.
[31] Factors Controlling Human EmbryonicStem Cell Differentiation
By MAYA SCHULDINER and NISSIM BENVENISTY
Introduction
Human embryonic stem (ES) cells are pluripotent cell lines derived fromthe inner cell mass (ICM) of blastocyst stage human embryos.1,2 Thesecells possess self-renewal capabilities, which can be preserved through tightregulation of their growth conditions. Such regulation enables these cells toproliferate indefinitely in culture. However, once the cells are allowed todifferentiate, they spontaneously develop into various cell types.3 Thedifferentiation process can be influenced, to some extent, by use of external
1J. A. Thomson, J. Itskovitz-Eldor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V. S. Marshall,
and J. M. Jones, Science 282, 1145 (1998).2B. E. Reubinoff, M. F. Pera, C. Y. Fong, A. Trounson, and A. Bongso, Nat. Biotechnol. 18,
399 (2000).3 J. Itskovitz-Eldor, M. Schuldiner, D. Karsenti, A. Eden, O. Yanuka, M. Amit, H. Soreq, and
N. Benvenisty, Mol. Med. 6, 88 (2000).
446 DIFFERENTIATION OF MONKEY AND HUMAN ES CELLS [31]
Copyright � 2003, Elsevier Inc.All rights reserved.
METHODS IN ENZYMOLOGY, VOL. 365 0076-6879/2003 $35.00
growth factors.4 The special properties and human origin of human ES celllines make them a unique model system for elucidating the processes of earlyembryogenesis and analyzing the effects of growth factors on early stem cellsin the developing human embryo. In addition, they are an invaluablebiotechnological and medical tool as they provide an unlimited cell sourcefor generating purified cells for cellular transplantation. More than twodecades ago, mouse ES cells were isolated,5,6 and research conducted onthem has provided invaluable protocols and insights into ES cell growth,differentiation and manipulation.7–10 The work conducted on human EScells is based on these tools and concentrates on special methods to controldifferentiation into desired tissues. This review focuses on methods todifferentiate human ES cells.
Spontaneous Differentiation of Human ES Cells
ICM cells possess the potential to develop into all embryonic cell types.Obviously, in order for proper differentiation to occur, each cell must havethe capacity to respond to an enormous network of signals which must workin tandem to control the cellular fate. These signals include soluble factors,cell surface molecules, and intrinsic control mechanisms such astranscription factors that work parallel to such physical mechanisms asgravity and pressure. Human ES cells are derived from this ICM populationand preserve their pluripotent nature. In addition to having the ability toreadily differentiate, they are expected to respond similarly to developmentalcues. Indeed, in the absence of necessary support from a feeder layer andspecific media additions, human ES cells spontaneously differentiate. Whendifferentiating in a monolayer, ES cells have been observed to form mainlyextra-embryonic cells. In order to produce a wide range of cellularphenotypes from the three embryonic germ layers, human ES cells wereallowed to aggregate into spheroid clumps termed embryoid bodies (EBs).3
4M. Schuldiner, O. Yanuka, J. Itskovitz-Eldor, D. A. Melton, and N. Benvenisty, Proc. Natl.
Acad. Sci. USA 97, 11307 (2000).5M. J. Evans and M. H. Kaufman, Nature 292, 154 (1981).6G. R. Martin, Proc. Natl. Acad. Sci. USA 78, 7634 (1981).7A. Bradley and E. J. Robertson, in ‘‘Current Topics in Developmental Biology,’’ Vol. 20,
p. 357. Yamada Science Foundation and Academic Press, Japan, 1986.8R. H. Lovell-Badge, in ‘‘Teratocarcinomas and Embryonic Stem Cells: A Practical Approach’’
(E. J. Robertson, ed.), p. 153. IRL Press, Washington DC, 1987.9E. J. Robertson, in ‘‘Teratocarcinomas and Embryonic Stem Cells: A Practical Approach’’
(E. J. Robertson, ed.), p. 71. IRL Press, Washington DC, 1987.10T. Burdon, I. Chambers, C. Stracey, H. Niwa, and A. Smith, Cell. Tiss. Org. 165, 131 (1999).
After several days as EBs various cell types appeared expressing markers ofthe three embryonic germ layers, and the human cells now responded to arange of extra-cellular signals that effect their differentiation capacity. It hasbeen shown that human ES cells and their differentiated derivatives expressa broad spectrum of receptors for growth factors.4 This is yet anotherindication of their ability to respond to a wide variety of signals. Afterformation of EBs, the cells readily differentiate into several cell types4 suchas muscle, bone, kidney, skin, liver, neurons,2,3 endothel,12 hematopoieticcells,3 pancreatic b cells13 and even functional cardiomyocytes3,14 (for adetailed description on the range of different cell types, see Table I). Thisprocess is not organized and does not lead to the formation of entire organs.Lack of proper gastrulation signals and organizer activity during theaggregation step are most likely responsible for this. Despite the lackof spatial organization, temporal cues do apparently exist as can beseen in mouse ES cells. During EB formation, the differentiatingcells sequentially activate tissue specific genes. For example, endothelialspecific transcripts15 or erythrocyte globins16 are transcribed in a mannerthat recapitulates their normal timed order of appearance during earlyembryonic development.
While EB formation is an important factor in influencing the formationof a variety of cell types, it is not necessarily essential for every type ofdifferentiation. Ectodermal cells, such as neurons may form in amonolayer.2 This is a likely consequence of their being the first differentiatedcells to form in the developing embryo and as such probably do not requireas many inductive signals.
Protocol for Formation of EBs
Human ES cells are usually grown on a layer of feeder cells, such asarrested mouse embryonic fibroblasts (MEFs) in order to retain their self-renewal and pluripotency properties. It is advisable, before beginning the
11C. L. Mummery, A. Feyen, E. Freund, and S. Shen, Cell Differ. Dev. 30, 195 (1990).12S. Levenberg, J. S. Golub, M. Amit, J. Itskovitz-Eldor, and R. Langer, Proc. Natl. Acad. Sci.
USA 99, 4391 (2002).13S. Assady, G. Maor, M. Amit, J. Itskovitz-Eldor, K. L. Skorecki, and M. Tzukerman,
Diabetes 50, 1691 (2001).14 I. Kehat, D. Kenyagin-Karsenti, M. Snir, H. Segev, M. Amit, A. Gepstein, E. Livne,
O. Binah, J. Itskovitz-Eldor, and L. Gepstein, J. Clin. Invest. 108, 407 (2001).15D. Vittet, M. H. Prandini, R. Berthier, A. Schweitzer, H. Martin-Sisteron, G. Uzan, and
E. Dejana, Blood 88, 3424 (1996).16M. H. Lindenbaum and F. Grosveld, Genes Dev. 4, 2075 (1990).
450 DIFFERENTIATION OF MONKEY AND HUMAN ES CELLS [31]
differentiation process, to transfer the cells for a single passage onto achemical matrix, such as 0.1% gelatin coated plates, in order to reduce thepercentage of MEFs present in the culture. Human ES cells are grown inmedium that contains: 400 ml KnockOutTM DMEM, 80 ml KnockOutTM
SR, 5ml (2 mM) glutamine, 50 �l (0.1 mM) b-mercaptoethanol, 5 ml (1�)nonessential amino acids stock, 2.5 ml (50 units/ml) penicillin (50 �g/ml)streptomycin, 1 ml (4 ng/ml) basic fibroblast growth factor (bFGF). Uponreaching confluence, cells should be dissociated from the plate using 0.1%Trypsin/1 mM EDTA. To form EBs the dissociated cells are placed intopetri dishes at a high concentration (approximately 107 cells per 10 cmplate). Bacterial petri dishes should be used to avert adherence of the cells tothe plate, thus allowing spontaneous aggregation (see Fig. 1B). EBs aregrown in the same medium as ES cells but without the addition of bFGF.After 2 days of incubation, clumps of cells form and the medium can bechanged for the first time. Medium is changed either by tilting the plate andaspirating approximately half of the media, or by gently transferring the
FIG. 1. Formation of embryoid bodies. The three stages of EBs development: I-Simple EB,
II-Cavitated EB, III-Cystic EB. Schematic protocol for preparing EBs either in suspension or by
use of the ‘‘hanging drop’’ method.
[31] DIFFERENTIATION OF HUMAN ES CELLS 451
EBs into conal tubes and allowing the clumps to settle (do not centrifuge).The medium is then aspirated and the EBs returned to the petri dish afterbeing resuspended in new growth media. Media should be changed every2 days in the manner described above. The EBs develop in three mainstages: 2 days following aggregation, small clusters, or simple EBs, arevisible (see Fig. 1A-I). A few days later the EBs appear dark in the center asthey start cavitating (see Fig. 1A-II). The final stage is characterized by theformation of cystic EBs which possess a fluid filled cavity and harbor manydifferentiated cell types (see Fig. 1A-III). The different sizes of EBs formedusing this method may effect their differentiation status. Thisobstacle can be overcome by determining a specific EB size using the‘‘hanging drop’’ method.17 This procedure involves placing drops ofcells onto a tissue culture plate lid. The lid is then placed back onto aphosphate buffered saline (PBS) filled plate to avoid drying up the samples(see Fig. 1B). In this way a specific amount of cells aggregate at the bottomof the drop and form homogeneous EBs. Drops should be 40 �l in size andcontain approximately 2000–4000 cells. No more than 25 drops should beplaced onto a 10-cm plate lid. This limit will ensure they do not merge. Aftertwo days, EBs should be carefully collected and placed in a petri dish.Growth should then be continued according to the first protocol.
Factors Effecting Differentiation of Human ES Cells
In the developing embryo, gastrulation is followed by formation of theneural tube. This initiates a cascade of inductive interactions betweendifferent cell types and germ layers and eventually leads to the formation ofthe complete organism. By placing ES cells in a specific embryonic locationadvantage may be taken of inductive signals that are important for directingdifferentiation into a particular tissue. Indeed, when this procedure wasperformed through injecting human ES cells in the vicinity of the chickneural tube, neuronal differentiation was observed.18 These signals can bepartially reconstructed in vitro by growing the cells on specialized feederlayers. As cell lines produced from different tissues secrete a variety ofsubstances into the growth media, they are expected to effect differentiationin a complex manner. Such matrices influence the stem cells not only viasecreted growth factors but also through their cell surface proteins.Specialized feeder layers have been used to direct differentiation of human
17V. A. Maltsev, J. Rohwedel, J. Hescheler, and A. M. Wobus, Mech. Dev. 44, 41 (1993).18R. S. Goldstein, M. Drukker, B. E. Reubinoff, and N. Benvenisty, Dev. Dyn. 225, 80 (2002).
452 DIFFERENTIATION OF MONKEY AND HUMAN ES CELLS [31]
ES cells to hematopoietic19 and cardiac muscle cells20 (see Table I). Thedrawback of such a system is that feeder layer cells must be eliminatedbefore therapeutic use for transplantation. Another disadvantage is thatthe actual signals required from the matrix remain unknown.These problems can be partially addressed by substituting the feeder layerwith better-defined conditions such as soluble growth factors and chemicalmatrices.
Soluble growth factors constitute the most studied group of effectors ofdifferentiation. There are approximately 20 families of cytokines found inhumans with roughly 100 different factors and their derivatives. The sheernumber of factors and possible combinations makes studying their effectsvery problematic. Furthermore, it is likely that human ES cells themselvessecrete growth factors. It is therefore virtually impossible to arrive at a trueanalysis of the effect of a single factor. Such obstacles not withstanding,single soluble growth factors have indeed been added to differentiating EScells. The results of this procedure indicated that each factor has a uniqueeffect on differentiation (see Table I). Thus, for example, activin A, inhibitedformation of most cell types analyzed, causing differentiation to mainlymesodermal tissues such as skeletal and cardiac muscle. Epidermal growthfactor (EGF) allowed formation of both ectodermal and mesodermal tissuesand enhanced expression of markers for skin cells in the culture. Whenneuronal growth factor (NGF) was present differentiation into cells of allthree germ layers occurred, with a more pronounced increase in the numberof neuronal cells. There was also an increase in expression of molecularmarkers for liver and pancreas cells derived from the endoderm.4 Anyobserved change in the cell types in culture could in principle occur as aresult of two separate mechanisms. The first is through ability to effectcellular fate decision so that cells differentiate into the desired path. Thesecond possibility is that the conditions cause positive and negative selectionoptimizing the growth of a specific cell type already present in the culture. Ifwe knew which mechanism was used by specific factors, we would be able todevelop better protocols: growth factors that direct differentiation maywork more efficiently if administered at early stages of the differentiationprocess. In the case of growth factors that work by selection, lateradministration following primary differentiation may be more effective. Inorder to administer growth factors optimally, their effective concentration
19D. S. Kaufman, E. T. Hanson, R. L. Lewis, R. Auerbach, and J. A. Thomson, Proc. Natl.
Acad. Sci. USA 98, 10716 (2001).20C. Mummery, D. Ward, C. E. van den Brink, S. D. Bird, P. A. Doevendans, T. Opthof,
A. Brutel de la Riviere, L. Tertoolen, M. van der Heyden, and M. Pera, J. Anat. 200, 233
(2002).
[31] DIFFERENTIATION OF HUMAN ES CELLS 453
levels should be taken into account. Many of the growth factors havecharacteristics of morphogens,21–23 and exert different effects according totheir concentration. Thus, several possible influences may emerge fromanalyses of a range of concentrations. Given these constraints it is hardlysurprising that no single factor tested until now has led to the formation of asingle cell type culture. Yet, the more complex the protocols and the greaterthe number of stages or growth factors used, the nearer this goal becomes.For example, neuronal cells are formed spontaneously after aggregationof EBs3 or when grown in a monolayer.2 When single growth factors suchas retinoic acid (RA) or NGF are added basal neuronal differentiationcan be enhanced from approximately 20% to approximately 50%.24
Methods utilizing manual isolation followed by growth factor combinationssuch as bFGF and EGF serve to further enhance these numbers.25
Finally, protocols that comprise several stages of growth factor admini-stration including epidermal growth factor (EGF), basic fibroblastgrowth factor (bFGF), platelet derived growth factor (PDGF), insulin likegrowth factor 1(IGF1), neurotrophin 3 (NT3) and brain derived neurotrophicfactor (BDNF), allow more homogeneous cultures to arise. These containclose to 100% neuronal cells that have also proved functional26 (see Table I).
Combining growth factors with chemical matrices may enable selectionof desired cell types and may even effect differentiation. The chemicalmatrices act through binding and activation of different cell surfacereceptors. Such combinations have been used in mouse ES cells to enhanceneuronal numbers in culture by utilizing laminin and polylysine, which arethe optimal matrices for obtaining neuronal proliferation in addition togrowth factors.27
If extra-cellular signals are not sufficient to achieve differentiation toa specific cell type, it is possible to directly activate the intra-cellularmolecular mechanisms controlling the cell fate. For example, in mouse EScells over-expression of HNF3b, a crucial transcription factor thatdetermines liver cell fate, caused endoderm formation and hepatocyte
21A. J. Durston, J. P. Timmermans, W. J. Hage, H. F. Hendriks, N. J. de Vries, M. Heideveld,
and P. D. Nieuwkoop, Nature 340, 140 (1989).22R. Dosch, V. Gawantka, H. Delius, C. Blumenstock, and C. Niehrs, Development 124, 2325
(1997).23N. McDowell, A. M. Zorn, D. J. Crease, and J. B. Gurdon, Curr. Biol. 7, 671 (1997).24M. Schuldiner, R. Eiges, A. Eden, O. Yanuka, J. Itskovitz-Eldor, R. S. Goldstein, and
N. Benvenisty, Brain Res. 913, 201 (2001).25B. E. Reubinoff, P. Itsykson, T. Turetsky, M. F. Pera, E. Reinhartz, A. Itzik, and T. Ben-Hur,
Nat. Biotechnol. 19, 1134 (2001).26M. K. Carpenter, M. S. Inokuma, J. Denham, T. Mujtaba, C. P. Chiu, and M. S. Rao, Exp.
Neurol. 172, 383 (2001).27M. Li, L. Pevny, R. Lovell-Badge, and A. Smith, Curr. Biol. 8, 971 (1998).
454 DIFFERENTIATION OF MONKEY AND HUMAN ES CELLS [31]
differentiation.28 Methods for genetically manipulating human ES cells haverecently been developed.29 This will allow similar procedures to be used inthese cells.
Protocol for Administration of Growth Factors
Growth factors are large proteins that may not diffuse freely in denselypacked EBs. If a growth factor does not reach every cell, analysis of its effectcan be problematic. As aggregation into EBs is an important step in theformation of the three germ layers, it is advisable that growth factors beadministered by means of a two-step protocol.4 During the first stage, EBsare formed over a period of 4 days. This period is sufficient for primarycellular interactions to occur within the EB. The second stage consists ofdissociation of the EBs into single cells and adding the growth factors to themonolayer. Dissociation can be performed by suspending the aggregates intrypsin for 10 min in 37�C whilst shaking the tube every 2 min to assuredispersal of the clumps. Single cells and remaining clumps should be platedon appropriate matrices such as: collagen, gelatin, fibronectin, or poly-lysine. The following day, after cells have adhered, growth factors are addedin the desired concentrations and refreshed every 2 days. Such a protocolfacilitates initial formation of the three embryonic germ layers as a result ofcellular interactions during the aggregation step, and homogeneousadministration of growth factors to the cell population.
Assaying for Cellular Differentiation
During differentiation of human ES cells, multiple cell types appear inparallel. It is therefore important to develop methods for monitoring adesired differentiation process. The ability to recognize a specific cell typemay also enable sorting into clean cultures, that is extremely important fortransplantation therapy or developmental research. By facilitating themonitoring of the differentiation process we can dramatically influence theability to optimize a differentiation protocol. Some cell types have beenstudied more than others or have easier features to analyze, but essentiallyany cell type can be identified provided there is enough information on itscharacteristics.
28M. Levinson-Dushnik and N. Benvenisty, Mol. Cell. Biol. 17, 3817 (1997).29R. Eiges, M. Schuldiner, M. Drukker, O. Yanuka, J. Itskovitz-Eldor, and N. Benvenisty,
Curr. Biol. 11, 514 (2001).
[31] DIFFERENTIATION OF HUMAN ES CELLS 455
Morphology
The most straightforward aspect of differentiation to document ismorphology by phase microscope. However, as most cells do not havedistinct morphological features this method is restricted to a few cell types.Examples of cells with easily identifiable morphologies are neurons withtheir distinct axonal outgrowths or muscle cells appearing with sincitia ofnuclei. In some cases, morphology is supplemented by function as in thecase of pulsating cardiomyocytes. However, even in the case of these cells,morphology is not sufficient to discriminate between cell subtypes, such asdopaminergic or serotonergic neurons. Where this type of analysis isrequired, a molecular procedure must be sought. Several cell types formunique tissue patterns when allowed to differentiate in three dimensions suchas in EBs or differentiated teratomas. Sectioning of these masses allows us tosee such structures as bone or gut. Human ES cell derived endothelial cellshave also been shown to form functional blood vessels when grown on amatrigel matrix or when forming teratomas.12
Molecular Markers
Molecular markers are commonly used to identify tissues that aredifficult to distinguish morphologically. They are also used for analysis ofcell subtypes. Each cell type, due to its unique function, has a specific geneexpression pattern that allows us to analyze for cell specific genes or cellspecific gene-combinations.
When assaying at the RNA level, it is possible to look at the entirepopulation of cells by using RT-PCR for specific markers, or DNAmicroarrays if a more complete expression profiling is required. Thesemethods document expression in large cell populations and thus do notallow us to estimate the number of cells that actually express a specificcellular marker. In situ hybridization can be used if quantification of specificcell types is required.
During differentiation most genes are regulated by transcription.Nevertheless, a more accurate analysis of gene expression is found at theprotein level. Assaying for protein presence can be performed on whole cellpopulations by Western blotting of protein extracts. Immunostaining ofsingle cells or tissue sections is a more commonly used method. In caseswhere the protein markers are extra-cellular they may easily be assayed forwhile retaining the cells viable through cell sorting methods. Such methodsinclude magnetic sorting using metal beads tied to secondary antibodies orfluorescence-activated cell sorting (FACS) utilizing fluorescent antibodies.These methods have the additional advantage of producing clean cell
456 DIFFERENTIATION OF MONKEY AND HUMAN ES CELLS [31]
cultures while also facilitating accurate estimation of cell proportions withinthe differentiating population. These methods are specifically advantageousfor assaying hematopoietic cells, as the surface molecules of these cells havebeen well characterized. In human ES cells, such methods have yielded purepopulations of CD34þ , hematopoietic progenitor cells.19 Protein modifica-tion or localization are sometimes required for proper function so that themere presence of proteins does not necessarily attest to their functionality.Ultimately, functional assays should be sought in order to assess correctdifferentiation.
An additional way of using tissue specific gene expression in order tofollow up on differentiated cell types can be performed by geneticallylabeling the cells. For example, transfection of marker genes such as b-galactosidase or green fluorescent protein (GFP) under a tissue specificpromoter, allows monitoring of cellular differentiation by following theexpression of the marker gene.27 Furthermore, GFP is an extremely efficienttool for sorting the cells utilizing a FACS. Similar methods using selectionmarkers, such as the neomycin resistance gene, allow growth of purified cellpopulations but make quantification more difficult.30,31 Extensive researchon transfection of mouse ES cells has showed that various chemical reagentsdo not introduce DNA into the cells efficiently, while physical pressure suchas electroporation or injection result in a much higher success rate. Asinjection requires special skills, the method of choice for geneticallymodifying mouse ES cells is electroporation.8 In human ES cells, it seemsthat electroporation is less efficient, whereas poly-cationic reagents havebeen shown to facilitate DNA introduction at a rate that is sufficiently highfor both transient and stable transfection procedures.29 In addition, it hasbeen shown that infection by lentiviral vectors is highly efficient in humanES cell lines.32 When stable integration of DNA in the genome is required,viral infection yields much higher success rates. However, it would seem thattransfection that does not introduce viral sequences has several advantagesincluding gaining easier approval of genetically manipulated cell lines foruse in clinical trials.
Protocol for Genetic Labeling of Human ES Cells
Human ES cells were shown to be efficiently transfected using the poly-cationic reagent ExGen (MBI-Fermentas).29 One day prior to transfection
30M. G. Klug, M. H. Soonpaa, G. Y. Koh, and L. J. Field, J. Clin. Invest. 98, 216 (1996).31S. Marchetti, C. Gimond, K. Iljin, C. Bourcier, K. Alitalo, J. Pouyssegur, and G. Pages,
J. Cell. Sci. 115, 2075 (2002).32A. Pfeifer, M. Ikawa, Y. Dayn, and I. M. Verma, Proc. Natl. Acad. Sci. USA 99, 2140 (2002).
[31] DIFFERENTIATION OF HUMAN ES CELLS 457
the cells are plated in 6-well tissue culture dishes at a density of 1.5–2�105
cells per single well. On the day of transfection, 1 ml of fresh media is placedin each well. A transfection master mix may be prepared. The procedure fora single well is to add 2 �g of DNA to 100 �l of 150 mM NaCl solution andvortexing. 6.6 �l of ExGen are then added to the DNA master mix (not thereverse order). This should be followed immediately by an additional vortexfor 10 sec to ensure homogeneous distribution of the reagents. The mix isleft at room temperature for 10 min to allow formation of transfectioncomplexes of the desired size. The transfection solution is now added toeach well while swirling to disperse evenly. The plates are centrifuged for5 min at 280g and then left for 30–45 min in an incubator. As the feederlayer cells are extremely sensitive to ExGen, it is recommended to rinsethe cells twice in PBS following the short incubation, and then add 2 ml offresh growth media to each well. For transient transfection the cellsare monitored between 24 and 72 hr after transfection. For stabletransfection it is extremely important that selection only beinitiated following passaging and disaggregation into single cells.Selection on the original colonies results in massive death of the colonycells, and does not allow propagation of single transfected cells. Followingpassaging into media containing the selection drug, resistant colonies arisefrom a single stable transfected cell approximately 10 days later. Whencolonies consisting of hundreds of cells become visible, each colony istransferred to a separate well of a 24-well tissue culture plates for expansion,freezing and analysis.
For stable transfection it is important to consider the growth of the cellsduring the selection process. Human ES cells grow on a feeder layer ofprimary MEF cells and not on immortalized cell lines such as STO that canbe more easily manipulated to express drug resistant markers. Yet, as withthe ES cells, MEF cells must also survive the selection procedure. It ispossible to bypass the need for drug resistant MEFs during the selectionprocess by growing the cells on a chemical matrix, such as matrigel, togetherwith conditioned media from the MEFs. Conditioned media is supple-mented with the necessary drug before addition to the ES cells, thusbypassing contact with the MEFs. This technique has been shown to sustainself-renewal for many passages.33 Another possibility is to produce theprimary fibroblasts from mice carrying resistance genes. Most geneticallymanipulated mice harbor the neomycin resistance gene. In addition,specialized mice strains carrying many drug resistances have been
33C. Xu, M. S. Inokuma, J. Denham, K. Golds, P. Kundu, J. D. Gold, and M. K. Carpenter,
Nat. Biotechnol. 19, 971 (2001).
458 DIFFERENTIATION OF MONKEY AND HUMAN ES CELLS [31]
developed. Examples include the DR4 mouse strain that can be utilized forneomycin, hygromycin, puromycin and 6TG selection.34
Functional Assays
In order to ascertain that proper differentiation has occurred it isnecessary to look at the functionality of the cell type that has beenproduced. This is, of course, even more important if the purpose of theresearch is to produce cells for transplantation medicine. Some tissuesneeded for transplantation such as cartilage may not necessitate anycomplex function other than the ability to form an intact tissue, whereasothers, such as neurons require very intricate functions that should becoordinated by proper connectivity. Methods used to assay in vitrofunctionality are tissue-specific. For example, functionality of neurons canbe determined by their ability to produce action potentials in responseto various signals. Methods for checking these parameters such aselectrophysiology and calcium imaging have also been used on neuronsderived from human ES cells. These analyses have shown that thedifferentiation protocol used facilitated formation of several types offunctional neurons such as GABAergic, glutamatergic, dopaminergic andcholinergic.26 The presence of functional cells does not necessarily indicatethat the response is physiological and as such will allow a proper,coordinated reaction if transplanted in vivo. With some tissues assessment iseven more straightforward. For example, the sole function of pancreatic bcells is to secrete insulin in a glucose dependent manner. When human EScells differentiated spontaneously into pancreatic b cells, it was suggestedthat these cells could produce elevated levels of insulin in media containinghigher glucose concentrations.13 Functionality of human ES cell derivedcardiomyocytes was assayed by observing pulsation3 or more accurately bypatch clamp electrophysiology that demonstrated rhythmic action potentialsin the contracting cardiomyocytes.20 Other options for monitoring humanES derived cardiomyocytes responsiveness to agonists and antagonist are:(1) External electrophysiology using a multi-electrode array that readsaction potentials from whole population or (2) intracellular calcium imagingfor single cells14 (for summary see Table I).
Tissue Integration In Vivo
It must be realized that not all methods for differentiation assessmentare interdependent: The fact that a cell type may have the right morphology
34K. L. Tucker, Y. Wang, J. Dausman, and R. Jaenisch, Nucleic Acids Res. 25, 3745 (1997).
[31] DIFFERENTIATION OF HUMAN ES CELLS 459
does not necessarily mean it expresses transcripts essential for its function.Moreover, correct gene expression does not always indicate protein presenceor function. The same may be said for tissue integration. Even in caseswhere cells differentiate and function properly, they may not have the abilityto integrate into the desired tissue due to lack of requisite cell surfacesignals. For this reason, after assessing functionality, it is important to alsoascertain whether integration occurs easily upon transplantation. Theultimate standard for analyzing differentiation would use transplantedcells in a diseased animal. This requires an animal model suffering fromorgan impairment or cellular dysfunction, and monitoring phenotypicchanges following transplantation of new cells. Improvement would indicatethat the cell is expressing all the necessary cellular markers needed forfunction and integration. Only after such analysis will it be possible todetermine the exact effect of various factors on human ES cells’differentiation. It is also important to perform this step beforetransplantation into human patients. So far studies of human ES celldifferentiation have only shown integration in neuronal systems. Cellsoriginating from human ES cells were indeed demonstrated to migrate andintegrate in animal brains.18,25,35 It is yet to be demonstrated that the in vivotransplanted cells function properly in the recipient animal. In order forsuch experiments to be performed and for medical transplantations tobecome feasible, methods for overcoming possible tissue rejection must befound.36,37
Conclusions
Over the past few years protocols have emerged for growing human EScells and differentiating them in vitro via the production of EBs. The use ofgrowth factors is important in order to achieve purified populations ofdifferentiated cells and to study the role of secreted molecules in earlyembryogenesis. The combination of different matrices, addition of growthfactors and transfection of transcription factors should yield more specificand mature cell types that must then be purified for further characterizationor medical use.
35S. C. Zhang, M. Wernig, I. D. Duncan, O. Brustle, and J. A. Thomson, Nat. Biotechnol. 19,
1129 (2001).36M. Drukker, G. Katz, A. Urbach, M. Schuldiner, G. Markel, J. Itskovitz-Eldor, B. Reubinoff,
O. Mandelboim, and N. Benvenisty, Proc. Natl. Acad. Sci. USA 99, 9864 (2002).37M. Schuldiner and N. Benvenisty, in ‘‘Recent Research and Developments in Molecular and
Cellular Biology’’ (S. Pandalai, ed.), Vol. 2, p. 223. Research Signpost, Trivandrum, 2001.
460 DIFFERENTIATION OF MONKEY AND HUMAN ES CELLS [31]
Acknowledgment
This research was partially supported by funds to N. B. from the Herbert Cohn Chair
(Hebrew University), by a grant from the Juvenile Diabetes Fund (USA), by a grant from the
Israel Science Foundation (grant no. 672/02-1) and by funds from the United States—Israel
Binational Science Foundation (grant no. 2001021). M. S. is a Clore fellow.
[31] Development of Cardiomyocytes fromHuman ES Cells
By IZHAK KEHAT, MICHAL AMIT, AMIRA GEPSTEIN, IRIT HUBER,JOSEPH ITSKOVITZ-ELDOR, and LIOR GEPSTEIN
Introduction
It is generally accepted that adult human cardiomyocytes are mostlyterminally differentiated and possess limited regenerative capacity followingsignificant cell losses such as those that occur during myocardial infarction.1
Cell transplantation is emerging as a novel strategy for myocardialregeneration, but has been hampered by the lack of a source for humancardiomyocytes.2 Similarly, the lack of a human cardiomyocyte cell line hassignificantly limited a variety of experimental procedures. The recentadvances in human embryonic stem cells (ES) research suggests a possiblesolution for this cell sourcing problem, since these unique cells can bepropagated in mass in vitro and coaxed to differentiate into the desiredlineage.3,4
Human ES cells are continuously growing cell lines of embryonic originthat were isolated from the inner cell mass of human blastocysts.3,4 Thesecells display the characterizing properties of ES cells, namely: derivationfrom the pre- or peri-implantation embryo, prolonged undifferentiatedproliferation under special conditions, and the capacity to form derivativesof all three germ layers.
One of the most fascinating and important aspects of human ES celllines is their ability to differentiate in vitro to advanced derivatives of all
1M. H. Soonpaa and L. J. Field, Circ. Res. 83, 15 (1998).2L. Reinlib and L. Field, Circulation 101, E182 (2000).3 J. A. Thomson, J. Itskovitz-Eldor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V. S. Marshall,
and J. M. Jones, Science 282, 1145 (1998).4B. E. Reubinoff, M. F. Pera, C. Y. Fong, A. Trounson, and A. Bongso, Nat. Biotechnol. 18,
399 (2000).
[31] CARDIOMYOCYTE DIFFERENTIATION FROM HUMAN ES 461
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