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Methods for Inducing Embryoid Body Formation- In Vitro Differentiation System of Embryonic Stem Cells

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  389J OURNAL   OF  B IOSCIENCE   AND  B IOENGINEERING ©2007, The Society for Biotechnology, JapanVol. 103, No. 5, 389–398. 2007DOI: 10.1263/jbb.103.389 REVIEW Methods for Inducing Embryoid Body Formation:  In Vitro  Differentiation System of Embryonic Stem Cells Hiroshi Kurosawa 1  Division of Medicine and Engineering Science, Interdisciplinary Graduate School of Medicine and Engineering,University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan 1 Received 9 January 2007/Accepted 13 February 2007 When cultured in suspension without antidifferentiation factors, embryonic stem (ES) cellsspontaneously differentiate and form three-dimensional multicellular aggregates called embryoidbodies (EBs). EBs recapitulate many aspects of cell differentiation during early embryogenesis,and play an important role in the differentiation of ES cells into a variety of cell types in vitro .There are several methods for inducing the formation of EBs from ES cells. The three basic meth-ods are liquid suspension culture in bacterial-grade dishes, culture in methylcellulose semisolidmedia, and culture in hanging drops. Recently, the methods using a round-bottomed 96-well plateand a conical tube are adopted for forming EBs from predetermined numbers of ES cells. For theproduction of large numbers of EBs, stirred-suspension culture using spinner flasks and bioreac-tors is performed. Each of these methods has its own peculiarity; thus, the features of formed EBsdepending on the method used. Therefore, we should choose an appropriate method for EB for-mation according to the objective to be attained. In this review, we summarize the studies on invitro  differentiation of ES cells via EB formation and highlight the EB formation methods recentlydeveloped including the techniques, devices, and procedures involved. [ Key    words: embryonic stem cells, embryoid body, embryoid body formation method, differentiation, culturevessel] The formation of embryoid bodies (EBs) is the principalstep in the differentiation of embryonic stem (ES) cells. EScells are pluripotent cell lines derived from blastocyst-stageearly mammalian embryos. Since the early 1980s, ES cellshave been isolated from the inner cell mass of a preimplan-tation blastocyst (1 –3). When maintained in the presence of  leukemia inhibitory factor (LIF) or in coculture with mouseembryonic fibroblasts (MEFs), ES cells retain their pluripo-tency and are capable of self-renewal (4, 5). ES cells con-tribute to the formation of all cell types in an organism in-cluding germ lines after being injected into recipient blasto-cysts. When cultured in the absence of LIF or MEF feederlayers, ES cells differentiate spontaneously, and then formthree-dimensional (3D) aggregates called EBs. This struc-ture facilitates multicellular interactions, in which cell-cellcontact exists and gap junctions may be established. An EB consists of ectodermal, mesodermal, and endodermal tissues, which recapitulate many aspects of cell differentiation dur-ing early mammalian embryogenesis and differentiate intoderivatives of all the three germ layers (6, 7). For this rea- son, EB formation has been utilized widely as a trigger of  in vitro  differentiation of both mouse and human ES cells.The most essential technique to form EBs is to culture EScells in suspension without antidifferentiation factors suchas LIF and feeder layer cells. Researchers have allowed EBformation according to a protocol that was designed to sat-isfy their purposes in a culture method selected from severaltypes. Three basic methods, namely, liquid suspension cul-ture in bacterial-grade dishes, culture in methylcellulosesemisolid media, and culture in hanging drops, are usuallyused for the formation of EBs to induce the formation of a variety of cell types from ES cells (8, 9). Other methods have also been employed instead of the three basic methods.Each of the methods has it own peculiarity, in which ES cells are cultured under various conditions; thus, the features of formed EBs are considered to be not homogeneous em-bryologically and morphologically. Strictly speaking, EBsare classified as simple EBs or cystic EBs according to thestage of differentiation (10 –12). In the case of mouse EScells, spherical ES cell aggregates with morula-like struc-tures formed in 2–4d in suspension culture are called sim-ple EBs. In the case of cystic EBs, a central cavity forms inEBs in 4–5d in suspension culture. Cystic EBs resemble an e-mail: kurohiro@yamanashi.ac.jpphone: +81-(0)55-220-8535fax: +81-(0)55-220-8536  KUROSAWAJ. B IOSCI . B IOENG .,390 embryo in the blastula or egg-cylinder stage, consisting of a double-layered structure with an inner ectodermal layer andan outer of endoderm enclosing the cavity. After 8–10d insuspension culture, cystic EBs expand to larger cystic struc-tures homologous to the visceral yolk sac of postimplan-tation embryos. It is suggested that these larger cystic EBsin later stages of in vitro  differentiation should be distin-guished from cystic EBs in 4–5d. In most reports, however,all EBs at various stages have been simply described as em-bryoid bodies (EBs) regardless of the method used for in-ducing EB formation, and the description on EB formationis too concise even though the differentiation status of EBsis markedly affected by the culture condition for EB forma-tion. This trend illustrates that the methodology of formingEBs is not become a big concern these days. Consideringthat EB formation remains important in the in vitro  differen-tiation of stem cells, more attention should be given to thetechniques, devices, and procedures used for EB formation.In this review, we deal in the details of culture methods forEB formation as in vitro  differentiation systems of ES cells. GENERAL APPROACHES TO INDUCE  IN VITRO DIFFERENTIATION OF ES CELLS Both mouse and human ES cells are routinely cultivatedin the presence of feeder layers (13 –15) to maintain theirstemness, that is, extensive regeneration potential and func-tional multilineage differentiation capacity. ES cells growon the feeder layers as colonies. Mouse ES cell colonies areenzymatically dissociated with trypsin to obtain a suspen-sion of single cells, which is then transferred for subcultureor differentiation culture. For human ES cells, small cell clumps are used in these operations. For mouse ES cells, LIF can substitute for feeder layers. However, LIF is not effec-tive for human ES cells.When factors that maintain the stemness of ES cells areremoved, ES cells spontaneously differentiate into deriva-tives of the three embryonic germ layers: the mesoderm, en-doderm, and ectoderm (16). Various protocols were estab- lished to promote the efficient and reproducible develop-ment of the cell type of interest. The following are basicstrategies to induce in vitro  differentiation of ES cells (17).EB formation: change in the culture condition from two-dimensional monolayer to three-dimensional structure. EScells dissociated from colonies are transferred into suspen-sion cultures, in which ES cells are allowed to aggregateand form spherical three-dimensional structures EBs (6, 8, 9, 18). The details on EB formation are described in a latersection. Modification of medium composition: nutrient restriction, reduction of serum concentration (19), and addition of a  growth factor having an impact on gene expression and cellproliferation. Forced proliferation generally results in cellslosing their differentiated phenotype, whereas suppressedproliferation results in the initiation of cell differentiation.The control of cell proliferation is related to direct ES cell differentiation towards specific lineages. Growth factors that affect proliferation and survival of specific cell types areoften added to a medium to promote differentiation (20 –24).The following are the typical growth factors often applied:basic fibroblast growth factor (bFGF), transforming growthfactor β  (TGF β ), activin-A, bone morphogenic protein 4(BMP-4), hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), nerve growth factor (NGF), and retin-oic acid (RA).Genetic manipulation of ES cells: forced expression of some transcription factors can direct differentiation of EScells toward specific lineages. For example, HOXB4 over-expression significantly enhances the hematopoietic poten-tial of mouse ES cells differentiated in vitro  (25). GATA-6 and GATA-4 expression in mouse ES cells induces theirdifferentiation into the extra-embryonic endoderm (26).GATA-4 overexpression enhances cardiogenesis and mark-edly increases the number of terminally differentiated beat-ing cardiomyocytes (27).Use of extracellular matrix (ECM) and signaling mole-cules: interaction between cells and ECM via integrins de-termines the expression of signaling molecules that affectES cell differentiation (28, 29). The ECM and integrins col-laborate to regulate gene expression associated with cell growth, differentiation, and survival. The developmental fate of differentiating stem cells depends on the complex of growth factors, signaling molecules, and the ECM proteinconstituting the developmental niche in which the stem cellsexist (30 –33). For example, cardiomyocytes are surroundedby a basement membrane consisting of type IV collagen,laminin, fibronectin, and several proteoglycans (34). TheECM applied to a culture system creates a microenviron-ment in vitro  similar to that in vivo . Coculture with stromal cells: stromal cell lines support ES cell differentiation. The stromal cell line OP9 (35), which isderived from a newborn mouse calvaria, supports hemato-genesis (36), and the bone-marrow-derived stromal cell lineST2 producing macrophage colony-stimulating factor (M-CSF) supports osteoclastogenesis from ES cells (37). Thepreadipose cell line PA6 (38) promotes neural differentia-tion of mouse ES cells (39), and also supports dopaminergic differentiation of human ES cells (40). All these approaches have specific advantages and disadvantages, and have beenused to generate a broad spectrum of cell types differenti-ated from ES cells (8). Among the above approaches, induc-ing EB formation is the most common method used for invitro  differentiation of both mouse and human ES cells. EBsare a powerful tool for studying the differentiation of EScells into specific and desired cell types (12). CULTURE METHODS FOR EB FORMATION The most reliable method for generating differentiatedcell types is the induction of EB formation, through which ES cells spontaneously develop into three-dimensional, mul- ticellular aggregates of differentiated and undifferentiatedcells. There are several methods to induce EB formation asschematically shown in Fig. 1. A common technique used isto simply deprive ES cells of contact with feeder cells, orfrom the presence of LIF, and culture ES cells under thecondition in which they are unable to adhere to the surfaceof the culture dishes (8). Three basic methods, namely, sus- pension culture in bacterial-grade dishes (Fig. 1a ), culture inmethylcellulose semisolid media (Fig. 1b), and culture in  METHODS FOR EMBRYOID BODY FORMATIONV OL . 103, 2007391 hanging drops (Fig. 1c), are usually used to induce ES cellsto form EBs (6, 9). In addition to above the three basicmethods, methods using a round-bottomed 96-well plate(Fig. 1d) and a conical tube (Fig. 1e) are used to form EBs from a predetermined numbers of ES cells. There is also a method for scalable production of EBs (Fig. 1f , g). In this review, the methods for EB formation are classified intofive categories, which are suspension culture in bacterial-grade dishes, methylcellulose culture (MC culture), hangingdrop culture (HD culture), suspension culture in low-adher-ence vessels, and spinner flask and bioreactor techniques forscalable production. Suspension culture in bacterial-grade dishes A bac-terial-grade dish, which is a nontreated polystyrene dishwith hydrophobicity, has been used for liquid suspensionculture of ES cells to induce EB formation (Fig. 1a ). In 1985, Doetschman et    al  . (41) developed a technique of forming EBs from ES cells in suspension culture using bac-terial-grade dishes (60mm or 100mm in diameter). Gener-ally, 10ml of ES cell suspension with a density of 10 3  –10 6 cells/ml is seeded into a 100-mm bacterial-grade dish.Seeded ES cells do not attach to the plastic surfaces of the bacterial-grade dishes, and they naturally stick to each other, and form aggregates without any shaking. Induction of EBformation in these bacterial-grade dishes has been utilizedto initiate the differentiation of ES cells into a variety of dif-ferentiated cell types. As for mouse ES cells, neural pro-genitors (42), vascular cells (43, 44), cardiomyocytes (45), chondrocytes (46), hepatic cells (47), insulin-producing cells (48, 49), and germ cells (50) are induced from EBs formed in the bacterial-grade dishes. As for human ES cells, neuralcells (7), hematopoietic cells (7), cardiomyocytes (7, 51), insulin-producing cells (52), and endothelial cells (53) are also induced from EBs. In bacterial-grade dishes, becauseES cells are allowed to aggregate spontaneously, the num-ber of cells incorporated into each aggregate varies. There-fore, the number and size of EBs formed in bacterial-gradedishes depend on the probability that ES cells encountereach other accidentally. Consequently, the size and shape of the resulting EBs tend to be heterogeneous (54). Heteroge-neously sized EBs will rapidly lose any synchrony in differ-entiation. It is necessary to form EBs in a uniform and re-producible manner with regulated homogeneity in morphol-ogy and differentiation status. To improve the homogeneityof EBs formed in bacterial-grade dishes, rotating suspen-sion culture was introduced (55). Bacterial-grade dishes (100mm in diameter; 10ml of cell culture per dish; initialcell density, 1 × 10 5 cells/ml) are rotated on a horizontallyrotating device at 50rpm. The use of this rotation culturesystem improved oxygen supply and enabled high-densityculture. This rotation culture system seems to be superior tostationary suspension culture in terms of the homogeneityof EBs formed, but it is not in general use yet. Methylcellulose culture MC culture was srcinallyemployed to form cell aggregates of a clonal srcin (56). When ES cells are seeded onto semisolid methylcellulosemedia, they tend to remain single, isolated by the matrix of methylcellulose, and these single ES cells will develop into aggregates (EBs). When a standard medium without methyl- cellulose is used, ES cells (or the resulting EBs) will ran-domly coalesce into very large structures. Therefore, MCculture allows reproducible formation of EBs from singleES cells. MC culture has been used for the study of hemato-poietic differentiation of ES cells (57 –60), because hemato-poietic cells can be efficiently generated in the differenti-ation system via EBs formed by MC culture. Usually, EScells are cultivated in 1% methylcellulose medium in bac-terial-grade dishes at a density of 1000–3000cells/ml. Wilesand Keller (57) have reported that hematopoietic cells are detected in more than 50% of all EBs formed by MC cul-ture. This high frequency might be due to a local accumula-tion of factors in the matrix of methylcellulose surroundingEBs. Potocnik et    al  . (60) developed a cell culture systemby which ES cells undergo hematopoietic differentiation ina low-oxygen (5%-O 2 ) atmosphere without additional exo-genous factors. After 15–20d of culture under these condi-tions, they detected lymphoid precursors. On the other hand,methylcellulose matrix disturbs the mass transfer of factorsadded during an experiment, and the handling of semisolidsolution by pipettes is not easy (61). A recent report com- paring the efficiency of hematopoietic differentiation showed  no differences between the three basic methods to induceEB formation, namely, suspension culture in bacterial-gradedishes, HD culture, and MC culture (18).Besides in hematopoietic differentiation, the MC cultureis also employed in the differentiation of endothelial cells(62) and in hematopoietic colony-forming assays (63, 64). Hanging drop culture HD culture (Fig. 1c) is the EB formation induction method that has been frequently andwidely used to differentiate ES cells into a variety of celltypes. Hanging drops provide ES cells a good environmentfor forming EBs. The rounded bottom of a hanging dropallows the aggregation of ES cells. The number of ES cellsaggregated in a hanging drop can be controlled by varyingthe number of cells in the initial cell suspension to be hungas a drop from the lid of a petri dish. Using the hanging dropmethod (HD method), therefore, we can reproducibly form homogeneous EBs from a predetermined number of ES cells. The following is the typical protocol of the HD method (9, 56, 65). (i) Plate 20–30- µ l drops containing 400–1000 EScells on the lid of petri dishes in regular arrays. A standard100-mm dish can accommodate about 30–40 drops. (ii) In- FIG.1.Schematic representation for vessels used in methods toform EBs from ES cells. When cultured in the absence of antidifferen-tiation factors (LIF and feeder layer cells), ES cells differentiate spon-taneously and form spherical three-dimensional aggregates called em-bryoid bodies (EBs).  KUROSAWAJ. B IOSCI . B IOENG .,392 vert the lid and place it over the bottom of a petri dish filledwith PBS to prevent the drops from drying out. When the lidis inverted, each drop hangs and the ES cells fall to the bot-tom of the drop. (iii) Incubate the petri dish with hangingdrops in an incubator for 2d. The ES cells aggregates into a single EB. (iv) Harvest EBs and subsequently transfer thesuspension of EBs into bacterial-grade dishes and cultivatethem for 3–5d. (In some cases, HD culture is conducted for5 d without culture in bacterial-grade dishes [66 –68].) (v)Plate 5- or 7-day-old EBs on gelatin-coated tissue cultureplates, or keep them in suspension for further differentia-tion.EBs formed by the HD method have been used to gener-ate a broad spectrum of cell types, including neuronal cells(69), lymphoid (70), hematopoietic cells (71), cardiomyo- cytes (66, 72, 73), smooth muscle cells (74, 75), chondro- cytes (76, 77), renal cells (78), adipocytes (79), hepatocytes (or hepatocyte-like cells) (67, 80), insulin-producing cells (68), and gametes (81). From these differentiated cell types reported, it appears that EBs formed by HD method tend tobe used in the differentiation of ES cells to mesodermal lin-eages.The disadvantages of the HD method are as follows: theliquid volume of a drop is limited to less than 50 µ l due tomaintaining hanging drops on the lid by surface tension,medium exchange for a drop is practically impossible, and a direct microscopic observation of forming EBs in drops isdifficult during cultivation. In a usual case, the HD methodconsists of two steps, namely, the aggregation of ES cells indrops and the maturation of cell aggregates to EBs in sus-pension culture using bacterial-grade dishes. Therefore, a series of steps of the HD method may be troublesome. Suspension culture in low-adherence vessels Suspen-sion culture using low-adherence vessels has been conducted  to form spherical cell aggregates from various cell types, forexample, spheroids of hepatocytes (82, 83), neurospheres of  neural stem cells (84, 85), and EBs of embryonal carcinoma (EC) cells (14, 56). In some of these, low-adherence culture vessels except for bacterial-grade dishes are adopted. A dishcoated with proteoglycan (82) and an uncoated dish withpositively charged surfaces (83) are used to form floating spherical aggregates of adult rat hepatocytes. Round-bottomed, low-adherence 96-well plates are among the tools for forming EBs with high uniformity (Fig. 1d). A known number of ES cells to form an EB can be seeded intoeach well. The low-adherence round-bottomed well concen-trates ES cells of known number to the extremity of the welland promotes cell-cell contact and cell aggregation. There-fore, round-bottomed, low-adherence 96-well plates make itpossible to investigate the effect of the number of seeded EScells on the differentiation of EBs. Ng et    al  . (86) developeda method by which a known number of human ES cells wascompulsorily aggregated by centrifugation to promote theformation of EBs of homogeneous size in 96-well plates.They reported that the number of ES cells seeded into eachwell affected hematopoietic differentiation in resulting EBs.Efficient blood formation requires an excess of 500 cells perwell, and optimum erythropoiesis was observed when 1000cells were seeded per well.Coating the plastic surfaces of 96-well plates with a reagent that prevents cell adhesion to the plastic surfaces,allows cells to aggregate and form an EB in a well withoutcentrifugation. A nonionic detergent, pluronic (F-127;Sigma), is employed as a coating reagent (18). A single ES cell is placed into each well of a 96-well plate coated with10% pluronic solution to prevent cell attachment. It was re-ported that individual ES cells can form EBs with an effi-ciency of at least 42 ± 9%. A phospholipid polymer, 2-meth-acryloyloxyethyl phosphorylcholine (MPC), is a very effec-tive reagent that prevents cells from adhering to plastic sur-faces of culture plates (87, 88). Koike et    al  . (87) obtained EBs with a high uniformity from a defined number of mouse ES cells in round-bottomed 96-well plates coated with MPC(commercially available as low-cell-binding plates; cat. no.145399; Nunc, Tokyo). In experiments in which 200–4000ES cells in a 200- µ l medium deposited per well, an EB wasspontaneously formed in each well by 5-d cultivation with-out centrifugation. The diameter of EBs formed from 1000seeded cells was about 600 µ m. The resulting EB from 1000cells per well was morphologically preferable and effi-ciently differentiated into cardiomyocytes. The use of 96-well plates coated with MPC makes it possible to performmicroscopic observation of a specific EB selected as the ob- servation target during cultivation for EB formation. Figure 2 shows a typical EB formation.A polypropylene 1.5-ml conical tube with a screw cap isappropriate for EB formation from a large defined numberof ES cells (Fig. 1e). One milliliter of mouse ES cell sus- pension containing 2 × 10 4 cells is deposited into the conicaltube and the screw cap is loosely closed for oxygen supply.After 5d of cultivation, a large EB is formed in the conicaltube; cardiomyocytes (89) and hepatocyte-like cells (90) are induced from EBs by subsequent cultures for further differ-entiation.In EB formation using low-adherence vessels (round-bot-tomed 96-well plates and conical tubes), an EB can be com-posed of a predetermined number of ES cells per well, as inthe HD method. The use of low-adherence vessels also en- FIG.2. Typical process of EB forming from 1000 ES cells in a well of a round-bottomed 96-well plate coated with 2-methacryloyloxy-ethyl phosphorylcholine (MPC). Microscopy images show the eventsin the same field of vision. Scale bars correspond to 100 µ m.
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