Derivation of Mesenchymal Stem Cells from Human Embryonic Stem Cells

In the early to mid 1970’s, hallmark studies were published demonstrating the ability to isolate and derive adult fibroblast cell colonies from the bone marrow stroma and the spleen (Friedenstein et al., 1970; Friedenstein and Kuralesova, 1971). These fibroblast-like cells, later termed bone marrow stromal cells (BMSCs) or bone marrow-derived mesenchymal stem cells (MSCs), were shown to proliferate in culture, to continually grow upon passaging while maintaining stable karyotypic characteristics, and were comprised of cells that had multipotent potential to differentiate along multiple mesenchymal cell lineages such as bone, cartilage, fat and could support hematopoietic stem cell (HSC) differentiation (Bab et al., 1984; Bab et al., 1986; Friedenstein et al., 1970; Friedenstein et al., 1974b). Numerous studies spurred from these findings, which also led researchers in this area to explore the functions of these cells in vitro and in their normal microenvironment. Bone marrow stromal cells were transplanted in vivo to determine if they had the ability to re-establish the marrow microenvironment, and it was reported that the ex vivo expanded stromal cells did indeed restore the hematopoietic niche within the bone marrow (Friedenstein et al., 1974a). These experiments further developed the hypothesis that within the bone marrow stroma resided a heterogeneous mixture of cells that function as a repository of progenitors, known as MSCs, that may migrate out of their stem cell niche in response to disease, injury, and aging. Therefore, extensive investigation into the identification of MSCs and their utility for cell-replacement therapies were the basis for a new emerging field known as tissue engineering (Ashton et al., 1980; Bab et al., 1986; Owen and Friedenstein, 1988; Beresford, 1989; Jaiswal et al., 1997; Krebsbach et al., 1999). In the 1980’s and 1990’s, many groups further demonstrated that culture-adherent MSCs present in the marrow stroma were capable of differentiation into bone, cartilage, muscle, tendon, and fat for multiple species such as canine, chicken, rabbit, rat, and mouse (Jaiswal et al., 1997). Using the expertise gained from these culture systems, MSCs were then isolated and propagated from human adult bone marrow (hMSCs) (Bab et al., 1988); (Krebsbach et al., 1997). Human MSCs were then used with site-specific delivery vehicles to repair bone, cartilage, and other connective tissues (Haynesworth et al., 1992a; Haynesworth et al., 1992b). Additionally, a series of monoclonal antibodies were developed to identify characteristic surface markers on hMSCs, which would prove to be beneficial to researchers interested in not only identifying MSCs, but also subpopulations of osteoprogenitor cells


Introduction
In the early to mid 1970's, hallmark studies were published demonstrating the ability to isolate and derive adult fibroblast cell colonies from the bone marrow stroma and the spleen (Friedenstein et al., 1970;Friedenstein and Kuralesova, 1971). These fibroblast-like cells, later termed bone marrow stromal cells (BMSCs) or bone marrow-derived mesenchymal stem cells (MSCs), were shown to proliferate in culture, to continually grow upon passaging while maintaining stable karyotypic characteristics, and were comprised of cells that had multipotent potential to differentiate along multiple mesenchymal cell lineages such as bone, cartilage, fat and could support hematopoietic stem cell (HSC) differentiation (Bab et al., 1984;Bab et al., 1986;Friedenstein et al., 1970;Friedenstein et al., 1974b). Numerous studies spurred from these findings, which also led researchers in this area to explore the functions of these cells in vitro and in their normal microenvironment. Bone marrow stromal cells were transplanted in vivo to determine if they had the ability to re-establish the marrow microenvironment, and it was reported that the ex vivo expanded stromal cells did indeed restore the hematopoietic niche within the bone marrow (Friedenstein et al., 1974a). These experiments further developed the hypothesis that within the bone marrow stroma resided a heterogeneous mixture of cells that function as a repository of progenitors, known as MSCs, that may migrate out of their stem cell niche in response to disease, injury, and aging. Therefore, extensive investigation into the identification of MSCs and their utility for cell-replacement therapies were the basis for a new emerging field known as tissue engineering (Ashton et al., 1980;Bab et al., 1986;Owen and Friedenstein, 1988;Beresford, 1989;Jaiswal et al., 1997;Krebsbach et al., 1999). In the 1980's and 1990's, many groups further demonstrated that culture-adherent MSCs present in the marrow stroma were capable of differentiation into bone, cartilage, muscle, tendon, and fat for multiple species such as canine, chicken, rabbit, rat, and mouse (Jaiswal et al., 1997). Using the expertise gained from these culture systems, MSCs were then isolated and propagated from human adult bone marrow (hMSCs) (Bab et al., 1988); (Krebsbach et al., 1997). Human MSCs were then used with site-specific delivery vehicles to repair bone, cartilage, and other connective tissues (Haynesworth et al., 1992a;Haynesworth et al., 1992b). Additionally, a series of monoclonal antibodies were developed to identify characteristic surface markers on hMSCs, which would prove to be beneficial to researchers interested in not only identifying MSCs, but also subpopulations of osteoprogenitor cells (Haynesworth et al., 1992a;Haynesworth et al., 1992b); (Gronthos et al., 1999). Simultaneously, Caplan et al. used the embryonic chick limb bud mesechymal cell culture system as an assay for the purification of inductive factors in bone to further develop the technology for isolating, expanding, and preserving the stem cell capacity of adult human bone marrow-derived mesenchymal stem cells (Caplan, 2005). With this newly acquired knowledge and the emerging technologies in biomedical engineering, hMSCs became the principle cell source for cell-based pre-clinical bone tissue engineering studies. Currently, substantial advances have been made to address clinical needs for regeneration of damaged or diseased tissues. The three main approaches of cell-based clinical therapies that employ the use of hMSCs are: 1) from a tissue engineering standpoint where cells are incorporated into 3D biomaterial scaffolds for the replacement of tissue in vivo, 2) from a cell replacement therapy standpoint where allogeneic donor cells are used to replace ablated tumors and diseased cells; and 3) from a inductive standpoint where cells provide cytokine and growth factor cues that stimulate host reparative events and inhibit degenerative events (Caplan, 2005). Thus, clinical protocols were developed to establish that autologous hMSCs could be safely implanted back in order to reconstitute the marrow microenvironment for breast cancer and osteogenesis imperfecta (OI) patients following chemotherapy treatment (Koc et al., 2000;Horwitz et al., 2002). Additionally, hMSCs have been shown to have immunomodulatory effects and could induce immune suppression in patients (Le Blanc and Pittenger, 2005); (Aggarwal and Pittenger, 2005). Although the use of hMSCs has been successfully used in some cases, there are challenges that scientists and clinicians must overcome before the transplantation of these cells is incorporated into routine clinical practice. Specifically, the classic method to isolate MSCs from bone marrow relies on their capacity to adhere to plastic, their resistance to trypsinization during passaging, and proliferation in growth medium containing serum (Olivier et al., 2006). However, cell availability is greatly limited with this method because MSCs are present at low concentrations in the marrow, occurring at less than 1 in 100,000-500,000 nucleated cells (Caplan, 2005). Also, the availability of tissues for their isolation remains limiting and requires invasive procedures that may cause severe donor site morbidity. Therefore, an alternative source for generating MSCs can be found in human embryonic stem cells (hESCs) (Thomson et al., 1998). Human ESCs are an alternative source for generating MSCs due to the fact that they can theoretically be expanded infinitely and also because using these cells would eliminate the need for invasive cell harvesting techniques. Host immune rejection could also be circumvented by the use of autologous hESCs generated from nuclear transfer or from immune compatible allogeneic hESCs. Derivation of mesenchymal stem cells from human ES cells will further the understanding of the differentiation pathways and important cellular events that occur during early human development and could also have useful clinical applications. Because of the therapeutic potential, particularly in the areas of cell therapy and regenerative medicine, derivation of MSCs from hESCs (hESC-MSCs) has specific advantages over the current "gold standard" use of autologous and allogeneic adult hMSCs for bone tissue engineering. (Olivier et al., 2006).

Human embryonic stem cells
The major advancements in the area of stem cell culture, derivation, propagation, and differentiation paved the way for a pivotal discovery that was reported in a 1998 study from the University of Wisconsin. Thomson et. al. described the first successful isolation and long www.intechopen.com term sustained culture of a small cluster cells from the inner cell mass of four-day old embryos (Thomson et al., 1998). These cells, known as human embryonic stem cells (hESCs), represent a robust biologic tool and model system through which the scientific and medical communities will better understand human development, disease pathophysiology, organogenesis, and mechanisms for cellular differentiation; all of which will help develop and improve the field of regenerative medicine. These embryonic stem cells are derived by the selection and expansion of individual colonies rather than clonal expansion of a single cell. Human ESCs are pluripotent cells that are presumed to have virtually unlimited proliferation capacity in vitro, maintain normal karyotypic characteristics, sustain high levels of telomerase activity, and retain uniform undifferentiated morphology in prolonged culture (Thomson et al., 1998). In addition, hESCs have the ability to differentiate along the three embryonic germ layers in vivo as evidenced by teratoma formation after injection into severe combined immunodeficient (SCID) mice. The teratomas can contain gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). They have also been shown to express certain cell surface markers that are widely used to confirm pluripotency, such as stage-specific embryonic antigen SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. Oct-4, a transcription factor, has been identified as another key indicator of the undifferentiated state. To maintain their self renewal capacity, hESCs were originally cultured on mouse embryonic fibroblast (MEF) feeder layers and grown under serum-free conditions using serum replacement (SR) with supplements of basic fibroblast growth factor (bFGF). Under these culture conditions, hESCs have been passaged continuously and maintained pluripotency as well as a normal karyotype. However, it has been reported that hESCs have been successfully cultured with feeder cells of human origin, such as human bone marrow stromal cells (hBMSCs), human placental fibroblasts, human foreskin fibrobalsts (hFFs), feeders derived from hESCs, and on polymeric substrates in feeder free conditions (Cheng et al., 2003;Genbacev et al., 2005;Wang et al., 2005;Stojkovic et al., 2005;Hovatta et al., 2003;Villa-Diaz et al., 2010). In order to safely use hESCs in a clinical setting, it is imperative that that feeder-free and animal product-free culture conditions are explored further to overcome the risks of cross-transfer of pathogens from xenogeneic sources. The ability of hESCs to maintain an undifferentiated state indefinitely in culture and to differentiate into all cell types and tissues within the human body has created a high demand for research. Although the cells are of great scientific interest, progression of this type of research has been met with great controversy and resistance due to the ethical concern of destroying early human embryos for derivation of hESC lines (Knowles, 2004;Baschetti, 2005;Gruen and Grabel, 2006). Nevertheless, once the ethical concerns are abated through placement of the appropriate guidelines and policies on research, the hESC field will not only evolve, but will continue to rapidly progress toward monumental medical and scientific breakthroughs.

Human embryonic stem cell derived mesenchymal stem cells
The current major goal for hESC research in regenerative medicine is the controlled differentiation into specific progenitor cells for the purpose of replacing or regenerating damaged tissue. Therefore, the ability to obtain large quantities of multipotent cells from hESCs represents a challenge for cell based therapy and tissue engineering strategies that currently rely on human bone marrow stromal cells (hMSCs). Within the diverse population of hMSCs, there exist early progenitor mesenchymal stem cells capable of self-renewal and multi-lineage differentiation into cell types such as osteoblasts, chondrocytes, and adipocytes (Bianco et al., 2003;Wagers and Weissman, 2004). While hMSCs make a useful source of osteoprogenitor cells for tissue engineering strategies, they have limited proliferation and differentiation capacity. In contrast, hESCs which are able to proliferate indefinitely in vitro, represent a potentially unlimited source of mesenchymal stem cells. Recent studies demonstrate that the derivation of hESC-MSCS, mesenchymal precursors derived from hESCs, has been achieved via various isolation methods, and the generation of osteoblasts has been achieved in co-culture with primary bone derived cells (PBDs), in the presence of known osteogenic supplements, and in transwell co-culture with hBMSCs (Ahn et al., 2006;Cao et al., 2005;Duplomb, 2007;Karner et al., 2007;Karp et al., 2006;Sotille et al., 2003;Tong et al., 2007). Although the identification and characterization of hESC-MSCs has been reported, the data are quite vast and varied in terms of the derivation method, cell culture conditions, the mechanism of differentiation (epithelial-mesenchymal transition vs neural crest stem cell-mesenchymal differentiation), multilineage differentiation potential, and surface markers used to select for a pure mesenchymal stem cell subpopulation. As the field continues to evolve, careful attention should be placed on standardizing these parameters along clinical-grade good manufacturing practice (GMP guidelines). Through the isolation and identification of hESC-MSCs and the ability to produce a large supply of progenitor cells that can be genetically modified, the field hESC-MSC based tissue engineering and regenerative medicine strategies holds great promise.

Derivation methods
Thorough and extensive investigation into the definition, differentiation, and identification of mesenchymal stem cells has occurred over the last three decades. However, there are fundamental mechanistic and developmental concepts that remain poorly understood. The foundation laid by pioneers in the MSC field has provided current researchers with a breadth of knowledge to draw upon because the same fundamental questions are being investigated to identify the true ''MSC" from differentiating hESCs. Many investigators state that although MSCs isolated from the adult bone marrow have been shown to differentiate in vitro and in vivo, as well as have been successfully used in a clinical setting to repopulate the marrow environment in cancer patients, harvesting and utilizing adult hMSCs has disadvantages such as tissue availability, donor site morbidity, and host immune rejection (Caplan, 2005;Horwitz et al., 2002;Karp et al., 2006). Therefore, hESCs have been the topic of great discussion and interest as a potential repository of cells that can provide an unlimited number of specialized mesenchymal stem cells known as hESC-MSCs. Numerous isolation protocols have been reported describing successful derivation and differentiation of hESC-MSCs (Arpornmaeklong et al., 2009;Barberi et al., 2005;Brown et al., 2009;de Peppo et al., 2010a;de Peppo et al., 2010b;Evseenko et al., 2010;Karlsson et al., 2009;Karp et al., 2006;Kopher et al., 2010;Kuznetsov et al., ;Lian et al., 2007;Olivier et al., 2006;Smith et al., 2009;Trivedi and Hematti, 2007;Xu et al., 2004). One of the first reports of the derivation of a MSC-like progenitor population was in 2004, where fibroblast-like hESC derivatives were infected with a human telomerase reverse transcriptase (hTERT) retrovirus, as a result showed extended proliferative capacity, supported undifferentiated growth of hESCs as a feeder layer, and differentiated into osteoblasts (Xu et al., 2004). Following that study, another group reported the successful production of hESC-MSCs when cultured on murine OP9 stromal cells in the presence of heat-inactivated FBS, and www.intechopen.com indicated that the hESC-MSCs had a similar immunophenotype to hMSCs after flow cytometry was performed to purify the hESC-MSC population from the stromal cell feeder (Barberi et al., 2005). Another method for hESC-MSC production involved the use of spontaneously differentiated hESC colonies. The cells obtained became morphologically fibroblastic and homogenous after multiple passages, possessed a characteristic MSC immunophenotype, and supported hESC and hematopoietic progenitor cell growth (Olivier et al., 2006). Of particular importance, two reports showed the ability to reproducibly derive clinically compliant hESC-MSCs in a xeno-free environment where all contaminating animal-derived components were replaced with human-derived or recombinant components. Thus, they cultivated a hESC-MSC line suitable for clinical use ( (Karlsson et al., 2009;Lian et al., 2007). Other groups described similar findings, demonstrating that hESCs had the ability to reproducibly proliferate, differentiate, and commit to the mesodermal lineage in various cell culture conditions (both in monolayer and 3D) while retaining their multilineage differentiation potential and self renewal capacity, further demonstrating their high potential for tissue engineering applications (Arpornmaeklong et al., 2009;Brown et al., 2009;de Peppo et al., 2010a;de Peppo et al., 2010b;Evseenko et al., 2010;Kopher et al., 2010;Lian et al., 2007;Smith et al., 2009;Trivedi and Hematti, 2007). In summary, multiple approaches have attempted to achieve the most direct and efficient derivation of hESC-MSCs. A variety of studies have compared using the embryoid body (EB) step versus omitting this step, using multiple media formulations with and without serum, and using feeder-free cultures versus co-culture. These reports greatly contributed to the field, however, a concensus on the most appropriate method of isolation and culture is absolutely necessary to make hESC-MSC based therapies in a clinical setting a reality.

Osteoprogenitor cell differentiation from hESCs
Currently, there are major gaps in the knowledge about the growth factors and threedimensional milieu that influence and direct osteoblast differentiation. The generation of osteoprogenitors from hESC-MSCs has been shown to be successful as evidenced by osteogenic gene expression of runt-related transcription factor 2 (Runx2), collagen type 1A (Col1A1), bone-specific alkaline phosphatase (ALP), and osteocalcin (OCN); mineralized matrix confirmed by von Kossa and Alizarin Red staining; bone nodule formation in vitro; and bone formation in vivo in diffusion chambers and transplants to orthotopic sites (Duplomb, 2007). One of the first differentiation studies used cultured hESCs in the presence of defined osteogenic supplements for 21 days, and was able to demonstrate mineralization and induction of osteoblastic marker expression (Sotille et al., 2003). Human ESCs have been co-cultured with primary bone derived cells (PBDs) to induce osteoblast differentiation without the addition of exogenous factors, and cultured in vitro in the presence of known osteogenic factors without the embryoid body (EB) formation step -both studies confirming that hESCs have the capacity to differentiate into osteoblasts (Ahn et al., 2006;Karp et al., 2006). Whereas, other findings suggest that 12 day EB-derived hESC-MSCs are equally capable of undergoing multilineage differentiation in vitro (Cao et al., 2005). It has also been shown that hESC-MSCs can not only differentiate into functional osteoblasts and adipocytes and express markers characteristic of hMSCs, but they can also be successfullly transduced with an osteogenic lineage specific Col2.3-GFP lentivirus in order to track and isolate cells as they underwent differentiation. The transgene construct used has been shown to be a useful tool for studying hBMSC differentiation . When the hESC-MSCs began as pre-osteoblasts there was low GFP expression, however, increased GFP expression was www.intechopen.com detected after 28 days culture in osteogenic medium, suggesting that hES-MSCs differentiated into mature osteoblasts. The ability to track differentiation allowed the isolation of osteoprogenitor cells from the derived hESC-MSC population. These studies suggest that in particular, the osteoprogenitor populations derived from hESCs have tremendous potential, and can serve as a tool through which we can characterize early bone development and cellular behavior on bone-related biomaterials.

Gene transcription and proteomic array analyses
The therapeutic capacity of hESC-MSCs to treat a variety of diseases lies within their capability to different into numerous cell phenotypes to repair or regenerate tissues and organs. However, it remains to be determined if transplanted MSCs, whether of hESC or adult stem cell origin, contribute to and integrate within the majority of newly formed tissue, or perhaps via paracrine action mediate and stimulate host repair and regeneration. To that end, investigation into the therapeutic potential of the hESC-MSC paracrine proteome has been conducted. Within the study, defined serum-free culture medium was conditioned by hESC-MSCs and subsequently analyzed via multidimensional protein identification and cytokine antibody array analysis (Sze et al., 2007). The array data revealed over 200 unique gene products that play a role in biological processes such as metabolism, defense, response, and tissue differentiation including vascularization, hematopoiesis, and skeletal development. These processes and pathways are associated with numerous cellular processes that are activated to participate in injury, repair, and regeneration, as well as to facilitate immune cell migration to the site of injury, ECM remodeling, and increases in cellular metabolism (Sze et al., 2007). The identification of a large number of MSC secretory products that can act as paracrine modulators provides insight into the potential mechanism of action by which hESC-MSCs may participate in tissue repair and disease treatment. Another study investigated the gene expression profile of differentiating hESC-MSCs and reported that during derivation major transcriptional changes occurred, resulting in an expression profile very similar to that of hMSCs (de Peppo et al., 2010b). The major questions addressed were how the transcriptome may be affected by the hESC-MSC derivation process and whether hESCs and their MSC derivatives were distinct or equivalent to one another. The findings in the hESC-MSC population revealed a downregulation in pluripotency genes such as the OCT family of genes, NANOG, TDGF1, LIN28, GDF3, and ZIC3, down regulation in tumor development p53-associated genes LTBP2 and TFAP2A, up-regulation of mesodermal lineage commitment genes such as RUNX2, TGBR2, BMPR2, and TFAP2A, and up-regulation of genes supportive of craniofacial development and osteogenesis such as DLX1, DLX2, and MSX1. Lastly, and importantly, the immunological profile of hESC-MSCs displayed lower expression than hMSCs of HLA-ABC and HLA-DR, two markers characteristic of the inflammatory immune response. These findings suggest that the hESC-MSCs may be more immuno-privileged than hMSCs, thus another piece of evidence supporting the notion that hESC-MSCs represent a suitable alternative for cell transplantation therapies (Romieu-Mourez et al., 2007;de Peppo et al., 2010a).

Epithelial-mesenchymal transition
Cells within the body are derived from a single cell, with variations of cell phenotypes resulting from expression of a specific and defined transcriptome, thus further imparting diversity in cellular signaling and function. Epithelia are considered to be highly plastic during embryogenesis and have the ability to shuttle back and forth between mesenchyme www.intechopen.com and epithelia through the process know as epithelial-mesenchymal transition (EMT). It is one mechanism that gives rise to mesenchymal-like behavior to cells in numerous different settings (Kalluri, 2009). Historically, it has been proposed that epithelial cells have to be terminally differentiated to in order to perform defined functions involved in organ development. However, experimental evidence has suggested that epithelial cells can alter their phenotype based on the influence of microenvironment (Boyer et al., 2000). Therefore, EMT has been accepted as a mechanism by which fibroblasts and mesenchymal cells are formed in injured tissues. In the adult, the process of EMT occurs during tissue regeneration and wound healing by facilitating mesenchymal cell migration to invade surrounding tissues. This was described as one of the three EMT subtypes that occurs, and is also suggested to be an underlying mechanism for derivation of hESC-MSCs (Zeisberg and Neilson, 2009;Ullmann et al., 2007). It has been reported that hESCs grown in monolayer in feeder-free conditions, without MEFs or other supporting cells, form uniform sheets of epithelial cells after removal from standard feeder culture systems (Boyd et al., 2009;Ullmann et al., 2007). The uniform epithelial sheets exhibit characteristic mesodermal gene expression patterns that appear to undergo EMT that results in a highly proliferative population of cells that over time become uniformly homogenous with a mesenchymal stem cell morphology. It is in fact these homogenous cells that many researchers identify as hESC-MSCs, which have the ability to differentiate along multiple mesenchymal cell lineages in vitro. More specifically, these studies find that the hESCs that underwent mesenchymal differentation in monolayer culture were over 80% positive for E-cadherin, a characteristic epithelium marker, and maintained expression while cell morphology changed. Additionally, the cells that were undergoing apparent EMT were positive for the characteristic markers such as CD73, CD90, CD 105 and CD166, and negative for CD31, CD34, CD45, CD133 and CD146, further confirming the formation of a mesenchymal progenitor cell population (Boyd et al., 2009). The key significance of these studies is the finding that hESCs are behaving in culture in a manner similar to that of normal embryogenesis, thus underscoring the importance of using hESCs as a tool for better understanding overall human development.

Tissue engineering strategies for human clinical applications
A major challenge for using stem cells in a clinical setting is the need to identify an ideal stem cell candidate that is multipotent while retaining its self-renewal capacity. Although hMSCs make a useful source of progenitor cells for tissue engineering strategies, as evidenced by their multipotent potential and immunosuppressive characteristics, their limited proliferative and differentiation capacity represent an obstacle for therapeutic application. In contrast, hESCs with their ability to proliferate indefinitely in vitro and multi lineage differentiation capacity represent an unlimited source of progenitor cells, specifically, mesenchymal progenitor cells. Therefore, it is necessary to establish clinicalgrade GMP protocols for the derivation, identification, and isolation of hES-MSCs, to produce large quantities of genotypically homogenous progenitor cells that can be modified, and to fully characterize these cells for tissue regeneration strategies. Tissue engineering is an emerging field of research aimed at regenerating functional tissues by combining cells with a supporting substrate or biomaterial that possesses design characteristics that deliver progenitor cells and important signalling molecules in a spatially and temporally controlled manner, while promoting vascularization and tissue invastion into the interior of the scaffold. Ideally, biomimetic scaffolds designed for hESC-MSC based tissue engineering strategies would contain inductive signaling cues for proliferation and differentiation, possess composite material properties that conferred the ability to generate multi-layered hybrid tissues, and have tunable three-dimensional geometrical architecture that appropriately restores form and function to anatomical defects or diseased tissues. Within the hESC field, the use of 3D scaffolds has been employed in only a few reports (Arpornmaeklong et al., 2009;Ferreira et al., 2007;Kaufman et al., 2010;Levenberg et al., 2003;Kim et al., 2007;Kuznetsov et al., 2010;Smith et al., 2009). Investigators have used collagen scaffolds for hepatocye differentiation, and porous polylactic/polyglycolic biomaterial sponges to direct neural, chondrogenic, or hepatocytic lineages (Levenberg et al., 2003). While other studies have shown that 3D porous alginate scaffolds to provide a conducive environment for generation of well-vascularized embryoid body derived hESCs (Ferreira et al., 2007). Within the bone tissue engineering field, the use of architecturally designed scaffolds with hESC-MSCs is seen even less frequently. It has been reported that hESC-MSCs were capable of forming bone tissue in vivo when implanted subcutaneously after 8 weeks in the presence of BMP-2 (Kim et al., 2007). In 2009, Arpornmaeklong et al. reported the influence of composite collagen scaffolds on the osteogenic differentiation of hESC-MSCs in vitro as indicated by osteogenic gene induction, increased ALP activity, and the presence of mature bone ECM proteins; all of which are characteristic of the osteoblast phenotype. From an in vivo standpoint, enriched osteoprogenitor cells were encaspulated in fibrin gels mixed with ceramic particles and implanted in a rat calvarial defect model. After six weeks, the identification of transplanted hESC-MSCs in newly formed bone verified the role that MSCs derived from hESCs played in the bone regeneration process (Arpornmaeklong et al., 2009). Another study demonstrated that hESC-MSCs can form mineralized tissue in vitro when cultured on 3D nanofibrous polylactic acid (PLLA) in the presence of BMP-7, illustrating the capability of hESC-MSCs to differentiate in 3D culture for bone regeneration purposes (Smith et al., 2009). Most recently, a comprehensive study investigated multiple media formulations and cell culture conditions for efficient derivation of a homogenous hESC-MSC population. To determine their in vivo osteogenic potential, cells were implanted up to 16 wks with biphasic ceramic particles and histology revealed cells of human origin were embedded with the bone, including broad areas of multiple intertwining trabeculae (Kuznetsov et al., 2010). It is hypothesized that the hESC-MSCs not only require a 3D biomaterial, but also inductive cues. This suggests that for tissue formation, hESCs may require additional biological cues such as pro-osteogenic factors for attachment, proliferation, and directed differentiation on biomaterials. For bone formation specifically, hESCs may require an osteoconductive biomaterial with not only the appropriate scaffold architecture, but one that also can associate cellular and molecular elements to increase cellular response to the biomaterial.

Conclusion
Human ESC research is a rapidly developing field, and has the potential to impact the medical and scientific community immensely. It is vitally important that we continue to explore hESC biology in order to realize the potential of hESCs to cure diseases. The derivation of mesenchymal stem cells from human embryonic stem cells is an area of active investigation in that hESC-MSCs potentially offer insight into embryonic mesodermal development events, as well as provide information about underlying differentiation mechanisms and signaling pathways that have been unclear heretofor. In addition to elucidating the mechanisms by which hESC-MSCs differentiate, it is equally important to www.intechopen.com better understand how the 3D biomaterial microenvironment can be manipulated to direct and control this process. In general, stem cell research advances the knowledge and understanding of how an organism develops and how progenitor cells migrate from the stem cell niche to the site of damaged or diseased tissue. To improve upon the overall quality of human health, scientists must continue to work collaboratively with clinicians to drive translational "bench-to-bedside" research. To this end, extensive investigation into the xeno-free derivation, robustness, and non-tumorigenic safety of hESC-MSCs will be absolutely necessary as the field progresses toward the realization of clinical tissue engineering and regenerative medicine therapies.