**2. Interactions with stem cells**

#### **2.1. Direct attachment between stem cells and engineered materials**

In tissue engineering, either natural or synthetic materials have been investigated for interac‐ tion with stem cells and to control their behavior [11]. The benefits to co-culture system include their ability to provide complicated signal to stem cells by physical attachment or chemical excretion. Compared to biological materials, natural or synthetic biomaterials, the former have poor mechanical properties and easily cause immune-response depending on the source of the materials; the later have wide diversity in properties that may be designed according to mechanics, chemistry, and degradation. The toxicity and a limited repertoire of cellular interactions are concerned.

As for the group of natural materials, collagen, matrigel, alginate, and hyaluronic acid (HA) have been used for stem cell researches. Collagen gels have been widely used in stem cell study, including mesenchymal stem cells (MSCs) [12,13] and ES cells [14]. Matrigel consists of a mixture of molecules derived from natural ECM and has been investigated for stem cell culture without feeder cells and inducing differentiation. An improved three-dimension (3D) and serum-free approach was established to differentiate hES cells into functional endothelial cells [16]. Alginate is a seaweed-derived polyanion that forms hydrogels through ionic crosslinking. ES cells have been induced in alginate hydrogels for a variety of applications, typically encapsulated stem cells for transplantation [15]. HA is a polysaccharide found in many tissues and has been modified to form hydrogels. HA hydrogels have been investigated for the culture and growth of undifferentiated human ES cells and MSCs [17, 18].

Synthetic materials were also widely used in stem cell cultures [19]. Materials that degrade through either hydrolytic or enzymatic mechanisms have been synthesized, and the advantage is the tenability and versatility of these physical properties. A hydroxyester has been exten‐ sively used in the field of tissue engineering, primarily due to their history of biocompatibility and use in medicine. One composition was seeded with human ES cells for the regeneration of numerous tissues, including vascular and neural structures [20].

#### **2.2. Secreted Soluble factors**

Cell and extracellular-matrix components in the stem-cell niche are relatively predictable, although the complexity and integration of these elements is far from known. Soluble mediators of cellular response would also be expected and a number of soluble factors such as growth factors and cytokines are important for stimulate and control the differentia‐ tion of stem cells. Adding relative molecules to the culture medium can induce the differentiation. For example, bone marrow stromal cells (BMSCs) have been directed into all kinds of cells [21]. Alternatively, it is advantageous to secrete the molecules directly from the specific cells. The molecules that can induce differentiation is various, such as basic fibroblast growth factor (FGF), members of the transforming growth factor family (TGF), small molecules such as retinoic acid. Molecule release is typically controlled through diffusion, degradation, or combination of factors. Multiple growth factors have been delivered from the same scaffold based on polymer degradation rates [22]. However, regards to co-culture system, the cells used as basement feeder open up the possibility to control molecule secretion for complex signaling cascades in stem cell differentiation, although the regulating mechanism is difficult to be cleared.

#### **2.3. Mechanical and shape cues to control stem cell differentiation**

The following section will review several important properties in the design co-culture system

In tissue engineering, either natural or synthetic materials have been investigated for interac‐ tion with stem cells and to control their behavior [11]. The benefits to co-culture system include their ability to provide complicated signal to stem cells by physical attachment or chemical excretion. Compared to biological materials, natural or synthetic biomaterials, the former have poor mechanical properties and easily cause immune-response depending on the source of the materials; the later have wide diversity in properties that may be designed according to mechanics, chemistry, and degradation. The toxicity and a limited repertoire of cellular

As for the group of natural materials, collagen, matrigel, alginate, and hyaluronic acid (HA) have been used for stem cell researches. Collagen gels have been widely used in stem cell study, including mesenchymal stem cells (MSCs) [12,13] and ES cells [14]. Matrigel consists of a mixture of molecules derived from natural ECM and has been investigated for stem cell culture without feeder cells and inducing differentiation. An improved three-dimension (3D) and serum-free approach was established to differentiate hES cells into functional endothelial cells [16]. Alginate is a seaweed-derived polyanion that forms hydrogels through ionic crosslinking. ES cells have been induced in alginate hydrogels for a variety of applications, typically encapsulated stem cells for transplantation [15]. HA is a polysaccharide found in many tissues and has been modified to form hydrogels. HA hydrogels have been investigated for the culture

Synthetic materials were also widely used in stem cell cultures [19]. Materials that degrade through either hydrolytic or enzymatic mechanisms have been synthesized, and the advantage is the tenability and versatility of these physical properties. A hydroxyester has been exten‐ sively used in the field of tissue engineering, primarily due to their history of biocompatibility and use in medicine. One composition was seeded with human ES cells for the regeneration

Cell and extracellular-matrix components in the stem-cell niche are relatively predictable, although the complexity and integration of these elements is far from known. Soluble mediators of cellular response would also be expected and a number of soluble factors such as growth factors and cytokines are important for stimulate and control the differentia‐ tion of stem cells. Adding relative molecules to the culture medium can induce the differentiation. For example, bone marrow stromal cells (BMSCs) have been directed into all kinds of cells [21]. Alternatively, it is advantageous to secrete the molecules directly from

**2.1. Direct attachment between stem cells and engineered materials**

and growth of undifferentiated human ES cells and MSCs [17, 18].

of numerous tissues, including vascular and neural structures [20].

to control stem cell differentiation.

120 Regenerative Medicine and Tissue Engineering

**2. Interactions with stem cells**

interactions are concerned.

**2.2. Secreted Soluble factors**

Using soft lithography techniques, the influence of spatially patterned adhesion molecules on cell differentiation was investigated, such as cell spreading and shape on MSC differen‐ tiation, through control the cellular cytoskeleton. MSCs tended to the differentiation of osteoblasts, when they were patterned on larger islands of adhesion ligands, which allowed for cell spreading. On the other hand, MSCs on smaller islands stayed rounded and differentiated into adipocytes [23]. This study indicated that stem cell commitment could be regulated by adhesion molecules and is a consideration in design for inducing the differentiation of stem cells.

During the last decades, much progress has been made in the molecular understanding of early differentiation in stem cells. For example, neural inducer molecules, such as chor‐ din, noggin, and retinoic acid (RA), were identified, and several intracellular mediators of neural differentiation have been characterized. Multiple-step method involving in embry‐ oid body (EB) formation followed by different combination of growth factors was the most common method for inducing differentiation of ES cells [24, 25]. Although the methods can produce a good proportion of different type of specific cells, it has several apparent problems. (i) it is difficult to analyze and control each regulatory step of differentiation in this multiple-step because EBs contains many different kinds of cells, including endo‐ derm, mesoderm, and ectoderm cells; (ii) growth factors have complicated function in vivo. For example, RA, a strong teratogen, is supposed to perturb neural patterning and neuronal identities in EBs as it does in vivo. RA treatment of early embryos causes suppression of forebrain development. It is therefore preferable to avoid RA treatment for therapeutic application unless RA induces the particular type of neurons of one's interest; (iii) in order to avoid infection and rejection, the serum of animal origin should be excluded; (iv) multiplestep method is cost-consuming.
