**Induce Differentiation of Embryonic Stem Cells by Co-Culture System**

Fengming Yue, Sakiko Shirasawa, Hinako Ichikawa, Susumu Yoshie, Akimi Mogi, Shoko Masuda, Mika Nagai, Tadayuki Yokohama, Tomotsune Daihachiro and Katsunori Sasaki

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56000

**1. Introduction**

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Stem cells, which are found in all multi-cellular organisms, can divide and differentiate into diverse special cell types and can self-renew to produce more stem cells. In mammals, two main broad types are included, such as embryonic stem (ES) cells and adult stem cells. The former are derived from the inner mass of blastocysts, and the latter have been found in various tissues from adult. In a developing embryo, stem cells can differentiate into all kinds of specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body.

ES cells, derived from the inner cell mass of pre-implantation embryos [1], can proliferate in culture and are able to give rise to all derivatives of the three primary germ layers: endoderm, mesoderm and ectoderm. In other words, they can develop into more than 200 cell types of the adult body when given stimulation for a specific cell type. The endoderm is composed of the entire gut tube and the lungs; the ectoderm gives rise to the nervous system and skin; and the mesoderm gives rise to muscle, bone, blood, and so on.

ES cells, being pluripotent cells, make them an excellent candidate as a source of functional differentiated cells for tissue replacement and regenerative medicine and after disease or injury. Using stem cell in regenerative therapy requires specific stimulation or signals for specific differentiation. If implanted directly, ES cells will randomly differentiate into many different types of cells and cause a teratoma eventually. ES cell researchers still face a few of

the hurdles, including differentiating ES cells into specific cells while avoiding transplant immno-rejection [2].

Till to date, mouse embryonic stem (mES) cells and human embryonic stem (hES) cells have been used in researches. They require very different environments in order to maintain an undifferentiatedstate.MouseES cells are culturedona layer ofgelatinas anextracellularmatrix and require the presence of feeder cells (STO or SNL) and leukemia inhibitory factor (LIF) [3]. Human ES cells are grown on a feederlayer of mouse embryonic fibroblasts (MEFs) and require the presence of basic fibroblast growth factor (bFGF or FGF-2) [4]. ES cells will rapidly differen‐ tiate without optimal culture conditions or genetic manipulation [5].

The multi-lineage differentiation potential of stem cells is not only an opportunity but also a challenge. An undesired cell type may lead to a pathophysiologic state or a non-functional tissue construct once that stem cells differentiate at the wrong time or place. In order to avoid such maladaptive responses, stem cells have evolved elaborate circuitry that triggers them to respond to differentiation cues only in an appropriate biological context. While most of researchers have been focusing on the role of soluble cues (e.g. growth factors and cytokines) in regulating stem cell differentiation, recent evidence demonstrated that the response to these stimuli are strongly modified by adhesive and mechanical cues, and that these microenviron‐ ment factors may be used explicitly to control stem cell differentiation in their own right[6]. With these advances in stem cell research, mimicking cellular microenvironment in vitro is becoming increasingly oriented toward to guide stem cell growth and differentiation.

In a living organism, cells are surrounded by peripheral other cells and embedded in an extracellular matrix (ECM) that defines the architecture, signaling, and biomechanics of the cellular microenvironment. As for stem cell, the word "niche" can be in reference to the in vivo and in vitro stem cell microenvironment. In architecture, the word niche refers to a recess, and in ecology it refers to a habitat where an organism can reside and reproduce. So, the grand position of the stem cell in popular concepts of science is appropriately humbled by the cells dwelling in a place where they might awaken with fleas. The concept of a niche as a specialized microenvironment housing stem cells was first proposed by Schofield, although experimental evidence was first provided by invertebrate models. In the gonads of Drosophila melanogaster and Caenorhabditis elegans, the germ stem cells reside at the distal end of a tapered structure, and have been shown to depend upon interactions with somatic cells so that stem-cell features could be maintained [7].

Stem cells are defined by their ability in complex multi-dimensional environment name as niche. Within the niche, several factors are important to regulate stem cell characteristics: (i) cell-cell interactions among stem cells; (ii) interactions between stem cells and neighboring differentiated cells; (iii) interactions between stem cells and extracellular matrix, adhesion molecules, soluble factors (growth factors, cytokines), oxygen tension, and other nature of the environment. Applying for regenerative medicine, specific differentiation of stem cells must be induced in vitro, and then specific graft with sufficient quantity and pure quality could be transplanted back into the patient. In order to archive this purpose, the researchers are trying to replicate the stem cell niche conditions in vitro. However, obviously, it is difficult to mimic the biological complexity of the native cell context in the laboratory under standard 2D culture conditions, since much of the complex interplay of mechanical and molecular factors present in vivo is absent in 2D culture status [8]. This is a major limitation to investigate cellular response in vitro. Therefore, we need generate a new culture system that would be "something between a culture dish and the cells", to represent cellular environment in a living organism and be more predictive of in vivo systems [9,10]. In particular, to stimulate stem cell potential and obtain biologically response in vitro, a new environment that are associated with their proliferation, differentiation, and assembly into tissues is desired. The researches should abide by the following premise: the function of the complicated factors is known to play a role during development or remodeling, and cellular responses to environment factors are predictable.

the hurdles, including differentiating ES cells into specific cells while avoiding transplant

Till to date, mouse embryonic stem (mES) cells and human embryonic stem (hES) cells have been used in researches. They require very different environments in order to maintain an undifferentiatedstate.MouseES cells are culturedona layer ofgelatinas anextracellularmatrix and require the presence of feeder cells (STO or SNL) and leukemia inhibitory factor (LIF) [3]. Human ES cells are grown on a feederlayer of mouse embryonic fibroblasts (MEFs) and require the presence of basic fibroblast growth factor (bFGF or FGF-2) [4]. ES cells will rapidly differen‐

The multi-lineage differentiation potential of stem cells is not only an opportunity but also a challenge. An undesired cell type may lead to a pathophysiologic state or a non-functional tissue construct once that stem cells differentiate at the wrong time or place. In order to avoid such maladaptive responses, stem cells have evolved elaborate circuitry that triggers them to respond to differentiation cues only in an appropriate biological context. While most of researchers have been focusing on the role of soluble cues (e.g. growth factors and cytokines) in regulating stem cell differentiation, recent evidence demonstrated that the response to these stimuli are strongly modified by adhesive and mechanical cues, and that these microenviron‐ ment factors may be used explicitly to control stem cell differentiation in their own right[6]. With these advances in stem cell research, mimicking cellular microenvironment in vitro is

becoming increasingly oriented toward to guide stem cell growth and differentiation.

In a living organism, cells are surrounded by peripheral other cells and embedded in an extracellular matrix (ECM) that defines the architecture, signaling, and biomechanics of the cellular microenvironment. As for stem cell, the word "niche" can be in reference to the in vivo and in vitro stem cell microenvironment. In architecture, the word niche refers to a recess, and in ecology it refers to a habitat where an organism can reside and reproduce. So, the grand position of the stem cell in popular concepts of science is appropriately humbled by the cells dwelling in a place where they might awaken with fleas. The concept of a niche as a specialized microenvironment housing stem cells was first proposed by Schofield, although experimental evidence was first provided by invertebrate models. In the gonads of Drosophila melanogaster and Caenorhabditis elegans, the germ stem cells reside at the distal end of a tapered structure, and have been shown to depend upon interactions with somatic cells so that stem-cell features

Stem cells are defined by their ability in complex multi-dimensional environment name as niche. Within the niche, several factors are important to regulate stem cell characteristics: (i) cell-cell interactions among stem cells; (ii) interactions between stem cells and neighboring differentiated cells; (iii) interactions between stem cells and extracellular matrix, adhesion molecules, soluble factors (growth factors, cytokines), oxygen tension, and other nature of the environment. Applying for regenerative medicine, specific differentiation of stem cells must be induced in vitro, and then specific graft with sufficient quantity and pure quality could be transplanted back into the patient. In order to archive this purpose, the researchers are trying to replicate the stem cell niche conditions in vitro. However, obviously, it is difficult to mimic the biological complexity of the native cell context in the laboratory under standard 2D culture

tiate without optimal culture conditions or genetic manipulation [5].

immno-rejection [2].

118 Regenerative Medicine and Tissue Engineering

could be maintained [7].

In our researches, we co-cultured ES cells with special cells to induce the specific differentia‐ tion. The co-culture system could supply stem cell for physical attachment (mechanical stretch), regulating signals, as well inducing factors such as cytokines (soluble or diffused). All these combined cues determined the differentiation of specific type of cells. The co-culture system recapitulated the combinations of parameters in the native environment to convert "collections of cells" into specific cell phenotypes. Hence, the design of co-culture system is necessarily inspired by stem cells research.

Co-culture system (ES cells and certain cell) supplied the physical, structural, and molecular factors to induce cellular differentiation. It opens several exciting possibilities: (i) establish functional implant which is suitable for transplantation and replace of degenerated tissues, (ii) investigate developmental processes and understand stem cell behavior in a native environ‐ ment; (iii) avoid using biomaterials in order to escape from immuno-rejection. A variety of parameters were outlined in Fig.1. It included co-culture system and engineered 2D culture environmentthatinfluencestemcellbehavior(e.g.,self-renewal,migration,anddifferentiation).

**Figure 1.** Manipulating the stem cell microenvironment in 2D and cell-stem cell co-culture system. Controllable pa‐ rameters include matrix properties, cell/cell contact and soluble factors which were secreted by specific feeder cells.

The following section will review several important properties in the design co-culture system to control stem cell differentiation.
