Embryoid body

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Phase image of EBs in suspension culture. Individual EBs are composed of approximately 1000 mESCs MESC EBs.jpg
Phase image of EBs in suspension culture. Individual EBs are composed of approximately 1000 mESCs
An embryoid body whose cells differentiated into enhanced green fluorescence protein-expressing spontaneously beating cardiomyocytes.

Embryoid bodies (EBs) are three-dimensional aggregates formed by pluripotent stem cells. These include embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC)

Contents

EBs are differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. They mimic the characteristics seen in early-stage embryos. They are often used as a model system to conduct research on various aspects of developmental biology. They can also contribute to research focused on tissue engineering and regenerative medicine.

Background

The pluripotent cell types that comprise embryoid bodies include embryonic stem cells (ESCs) derived from the blastocyst stage of embryos from mouse (mESC), [1] [2] primate, [3] and human (hESC) [4] sources. Additionally, EBs can be formed from embryonic stem cells derived through alternative techniques, including somatic cell nuclear transfer [5] [6] [7] or the reprogramming of somatic cells to yield induced pluripotent stem cells (iPS). [8] [9] [10] [11] Similar to ESCs cultured in monolayer formats, ESCs within embryoid bodies undergo differentiation and cell specification along the three germ lineages – endoderm, ectoderm, and mesoderm – which comprise all somatic cell types. [12] [13]

In contrast to monolayer cultures, however, the spheroid structures that are formed when ESCs aggregate enables the non-adherent culture of EBs in suspension, making EB cultures inherently scalable, which is useful for bioprocessing approaches, whereby large yields of cells can be produced for potential clinical applications. [14] Additionally, although EBs largely exhibit heterogeneous patterns of differentiated cell types, ESCs are capable of responding to similar cues that direct embryonic development. [15] Therefore, the three-dimensional structure, including the establishment of complex cell adhesions and paracrine signaling within the EB microenvironment, [16] enables differentiation and morphogenesis which yields microtissues that are similar to native tissue structures. Such microtissues are promising to directly [15] or indirectly [17] [18] repair damaged or diseased tissue in regenerative medicine applications, as well as for in vitro testing in the pharmaceutical industry and as a model of embryonic development.

Formation

EBs are formed by the homophilic binding of the Ca2+ dependent adhesion molecule E-cadherin, which is highly expressed on undifferentiated ESCs. [19] [20] [21] When cultured as single cells in the absence of anti-differentiation factors, ESCs spontaneously aggregate to form EBs. [19] [22] [23] [24] Such spontaneous formation is often accomplished in bulk suspension cultures whereby the dish is coated with non-adhesive materials, such as agar or hydrophilic polymers, to promote the preferential adhesion between single cells, rather than to the culture substrate. As hESC undergo apoptosis when cultured as single cells, EB formation often necessitates the use of inhibitors of the rho associated kinase (ROCK) pathway, including the small molecules Y-27632 [25] and 2,4 disubstituted thiazole (Thiazovivin/Tzv). [26] Alternatively, to avoid dissociation into single cells, EBs can be formed from hESCs by manual separation of adherent colonies (or regions of colonies) and subsequently cultured in suspension. Formation of EBs in suspension is amenable to the formation of large quantities of EBs, but provides little control over the size of the resulting aggregates, often leading to large, irregularly shaped EBs. As an alternative, the hydrodynamic forces imparted in mixed culture platforms increase the homogeneity of EB sizes when ESCs are inoculated within bulk suspensions. [27]

Formation of EBs can also be more precisely controlled by the inoculation of known cell densities within single drops (10-20 µL) suspended from the lid of a Petri dish, known as hanging drops. [21] While this method enables control of EB size by altering the number of cells per drop, the formation of hanging drops is labor-intensive and not easily amenable to scalable cultures. Additionally, the media can not be easily exchanged within the traditional hanging drop format, necessitating the transfer of hanging drops into bulk suspension cultures after 2–3 days of formation, whereby individual EBs tend to agglomerate. Recently, new technologies have been developed to enable media exchange within a modified hanging drop format. [28] In addition, technologies have also been developed to physically separate cells by forced aggregation of ESCs within individual wells or confined on adhesive substrates, [29] [30] [31] [32] which enables increased throughput, controlled formation of EBs. Ultimately, the methods used for EB formation may impact the heterogeneity of EB populations, in terms of aggregation kinetics, EB size and yield, as well as differentiation trajectories. [31] [33] [34]

Differentiation within EBs

Within the context of ESC differentiation protocols, EB formation is often used as a method for initiating spontaneous differentiation toward the three germ lineages. EB differentiation begins with the specification of the exterior cells toward the primitive endoderm phenotype. [35] [36] The cells at the exterior then deposit extracellular matrix (ECM), containing collagen IV and laminin, [37] [38] similar to the composition and structure of basement membrane. In response to the ECM deposition, EBs often form a cystic cavity, whereby the cells in contact with the basement membrane remain viable and those at the interior undergo apoptosis, resulting in a fluid-filled cavity surrounded by cells. [39] [40] [41] Subsequent differentiation proceeds to form derivatives of the three germ lineages. In the absence of supplements, the “default” differentiation of ESCs is largely toward ectoderm, and subsequent neural lineages. [42] However, alternative media compositions, including the use of fetal bovine serum as well as defined growth factor additives, have been developed to promote the differentiation toward mesoderm and endoderm lineages. [43] [44] [45]

As a result of the three-dimensional EB structure, complex morphogenesis occurs during EB differentiation, including the appearance of both epithelial- and mesenchymal-like cell populations, as well as the appearance of markers associated with the epithelial-mesenchymal transition (EMT). [46] [47] Additionally, the inductive effects resulting from signaling between cell populations in EBs results in spatially and temporally defined changes, which promote complex morphogenesis. [48] Tissue-like structures are often exhibited within EBs, including the appearance of blood islands reminiscent of early blood vessel structures in the developing embryo, as well as the patterning of neurite extensions (indicative of neuron organization) and spontaneous contractile activity (indicative of cardiomyocyte differentiation) when EBs are plated onto adhesive substrates such as gelatin. [13] More recently, complex structures, including optic cup-like structures were created in vitro resulting from EB differentiation. [49]

Parallels with embryonic development

Much of the research central to embryonic stem cell differentiation and morphogenesis is derived from studies in developmental biology and mammalian embryogenesis. [15] For example, immediately after the blastocyst stage of development (from which ESCs are derived), the embryo undergoes differentiation, whereby cell specification of the inner cell mass results in the formation of the hypoblast and epiblast. [50] Later on in postimplantation development, the anterior-posterior axis is formed and the embryo develops a transient structure known as the primitive streak. [51] Much of the spatial patterning that occurs during the formation and migration of the primitive streak results from the secretion of agonists and antagonists by various cell populations, including the growth factors from the Wnt and transforming growth factor β (TGFβ) families (Lefty 1, Nodal), as well as repressors of the same molecules (Dkk-1, Sfrp1, Sfrp5). [52] [53] [54] Due to the similarities between embryogenesis and ESC differentiation, many of the same growth factors are central to directed differentiation approaches.

In addition, advancements of EB culture resulted in the development of embryonic organoids (Gastruloids) which show remarkable parallels to embryonic development [46] [55] [56] [57] [58] [59] such as symmetry-breaking, localised brachyury expression, the formation of the embryonic axes (anteroposterior, dorsoventral and Left-Right) and gastrulation-like movements. [46] [55] [56] [57]

Challenges to directing differentiation

In contrast to the differentiation of ESCs in monolayer cultures, whereby the addition of soluble morphogens and the extracellular microenvironment can be precisely and homogeneously controlled, the three-dimensional structure of EBs poses challenges to directed differentiation. [16] [60] For example, the visceral endoderm population which forms the exterior of EBs, creates an exterior “shell” consisting of tightly connected epithelial-like cells, as well as a dense ECM. [61] [62] Due to such physical restrictions, in combination with EB size, transport limitations occur within EBs, creating gradients of morphogens, metabolites, and nutrients. [60] It has been estimated that oxygen transport is limited in cell aggregates larger than approximately 300 µm in diameter; [63] however, the development of such gradients are also impacted by molecule size and cell uptake rates. Therefore, the delivery of morphogens to EBs results in increased heterogeneity and decreased efficiency of differentiated cell populations compared to monolayer cultures. One method of addressing transport limitations within EBs has been through polymeric delivery of morphogens from within the EB structure. [61] [64] [65] Additionally, EBs can be cultured as individual microtissues and subsequently assembled into larger structures for tissue engineering applications. [66] Although the complexity resulting from the three-dimensional adhesions and signaling may recapitulate more native tissue structures, [67] [68] it also creates challenges for understanding the relative contributions of mechanical, chemical, and physical signals to the resulting cell phenotypes and morphogenesis.

See also

Related Research Articles

<span class="mw-page-title-main">Stem cell</span> Undifferentiated biological cells that can differentiate into specialized cells

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<span class="mw-page-title-main">Cellular differentiation</span> Developmental biology

Cellular differentiation is the process in which a stem cell changes from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Some differentiation occurs in response to antigen exposure. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly controlled modifications in gene expression and are the study of epigenetics. With a few exceptions, cellular differentiation almost never involves a change in the DNA sequence itself. However, metabolic composition does get altered quite dramatically where stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. Thus, different cells can have very different physical characteristics despite having the same genome.

<span class="mw-page-title-main">Gastrulation</span> Stage in embryonic development in which germ layers form

Gastrulation is the stage in the early embryonic development of most animals, during which the blastula, or in mammals the blastocyst, is reorganized into a two-layered or three-layered embryo known as the gastrula. Before gastrulation, the embryo is a continuous epithelial sheet of cells; by the end of gastrulation, the embryo has begun differentiation to establish distinct cell lineages, set up the basic axes of the body, and internalized one or more cell types including the prospective gut.

<span class="mw-page-title-main">Embryonic stem cell</span> Type of pluripotent blastocystic stem cell

Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells. Isolating the inner cell mass (embryoblast) using immunosurgery results in destruction of the blastocyst, a process which raises ethical issues, including whether or not embryos at the pre-implantation stage have the same moral considerations as embryos in the post-implantation stage of development.

<span class="mw-page-title-main">Oct-4</span> Mammalian protein found in Homo sapiens

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<span class="mw-page-title-main">Stem-cell line</span> Culture of stem cells that can be propagated indefinitely

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<span class="mw-page-title-main">Organoid</span> Miniaturized and simplified version of an organ

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<span class="mw-page-title-main">Induced pluripotent stem cell</span> Pluripotent stem cell generated directly from a somatic cell

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<span class="mw-page-title-main">Nodal homolog</span> Mammalian protein found in Homo sapiens

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Dental pulp stem cells (DPSCs) are stem cells present in the dental pulp, which is the soft living tissue within teeth. DPSCs can be collected from dental pulp by means of a non-invasive practice. It can be performed with an adult after simple extraction or to the young after surgical extraction of wisdom teeth. They are pluripotent, as they can form embryoid body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. DPSCs can differentiate in vitro into tissues that have similar characteristics to mesoderm, endoderm and ectoderm layers. They can differentiate into many cell types, such as odontoblasts, neural progenitors, osteoblasts, chondrocytes, and adipocytes. DPSCs were found to be able to differentiate into adipocytes and neural-like cells. DPSC differentiation into osteogenic lines is enhanced in 3D condition and hypoxia. These cells can be obtained from postnatal teeth, wisdom teeth, and deciduous teeth, providing researchers with a non-invasive method of extracting stem cells. The different cell populations, however, differ in certain aspects of their growth rate in culture, marker gene expression and cell differentiation, although the extent to which these differences can be attributed to tissue of origin, function or culture conditions remains unclear. As a result, DPSCs have been thought of as an extremely promising source of cells used in endogenous tissue engineering.

<span class="mw-page-title-main">Cell potency</span> Ability of a cell to differentiate into other cell types

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<span class="mw-page-title-main">P19 cell</span>

P19 cells is an embryonic carcinoma cell line derived from an embryo-derived teratocarcinoma in mice. The cell line is pluripotent and can differentiate into cell types of all three germ layers. Also, it is the most characterized embryonic carcinoma (EC) cell line that can be induced into cardiac muscle cells and neuronal cells by different specific treatments. Indeed, exposing aggregated P19 cells to dimethyl sulfoxide (DMSO) induces differentiation into cardiac and skeletal muscle. Also, exposing P19 cells to retinoic acid (RA) can differentiate them into neuronal cells.

Embryonic stem cells are capable of self-renewing and differentiating to the desired fate depending on their position in the body. Stem cell homeostasis is maintained through epigenetic mechanisms that are highly dynamic in regulating the chromatin structure as well as specific gene transcription programs. Epigenetics has been used to refer to changes in gene expression, which are heritable through modifications not affecting the DNA sequence.

Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor or unipotent – (iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation and are also called induced somatic stem cells.

Human engineered cardiac tissues (hECTs) are derived by experimental manipulation of pluripotent stem cells, such as human embryonic stem cells (hESCs) and, more recently, human induced pluripotent stem cells (hiPSCs) to differentiate into human cardiomyocytes. Interest in these bioengineered cardiac tissues has risen due to their potential use in cardiovascular research and clinical therapies. These tissues provide a unique in vitro model to study cardiac physiology with a species-specific advantage over cultured animal cells in experimental studies. hECTs also have therapeutic potential for in vivo regeneration of heart muscle. hECTs provide a valuable resource to reproduce the normal development of human heart tissue, understand the development of human cardiovascular disease (CVD), and may lead to engineered tissue-based therapies for CVD patients.

Directed differentiation is a bioengineering methodology at the interface of stem cell biology, developmental biology and tissue engineering. It is essentially harnessing the potential of stem cells by constraining their differentiation in vitro toward a specific cell type or tissue of interest. Stem cells are by definition pluripotent, able to differentiate into several cell types such as neurons, cardiomyocytes, hepatocytes, etc. Efficient directed differentiation requires a detailed understanding of the lineage and cell fate decision, often provided by developmental biology.

After the blastocyst stage, once an embryo implanted in endometrium, the inner cell mass (ICM) of a fertilized embryo segregates into two layers: hypoblast and epiblast. The epiblast cells are the functional progenitors of soma and germ cells which later differentiate into three layers: definitive endoderm, mesoderm and ectoderm. Stem cells derived from epiblast are pluripotent. These cells are called epiblast-derived stem cells (EpiSCs) and have several different cellular and molecular characteristics with Embryonic Stem Cells (ESCs). Pluripotency in EpiSCs is essentially different from that of embryonic stem cells. The pluripotency of EpiSCs is primed pluripotency: primed to differentiate into specific cell lineages. Naïve pluripotent stem cells and primed pluripotent stem cells not only sustain the ability to self-renew but also maintain the capacity to differentiate. Since the cell status is primed to differentiate in EpiSCs, however, one copy of the X chromosome in XX cells in EpiSCs is silenced (XaXi). EpiSCs is unable to colonize and is not available to be used to produce chimeras. Conversely, XX cells in ESCs are both active and can produce chimera when inserted into a blastocyst. Both ESC and EpiSC induce teratoma when injected in the test animals which proves pluripotency. EpiSC display several distinctive characteristics distinct from ESCs. The cellular status of human ESCs (hESCs) is similar to primed state mouse stem cells rather than naïve state.

<span class="mw-page-title-main">Gastruloid</span>

Gastruloids are three dimensional aggregates of embryonic stem cells (ESCs) that, when cultured in specific conditions, exhibit an organization resembling that of an embryo. They develop with three orthogonal axes and contain the primordial cells for various tissues derived from the three germ layers, without the presence of extraembryonic tissues. Notably, they do not possess forebrain, midbrain, and hindbrain structures. Gastruloids serve as a valuable model system for studying mammalian development, including human development, as well as diseases associated with it. They are a model system an embryonic organoid for the study of mammalian development and disease.

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