Pluripotent Adult Stem Cells: A Potential Revolution in Regenerative Medicine and Tissue Engineering

Stem cells are undifferentiated cells defined by their abilities to self-renew and differentiate into mature cells. Stem cells found in fully developed tissues are defined as adult stem cells. The function of adult stem cells is the maintenance of adult tissue specificity by homeostatic cell replacement and tissue regeneration (Wagers and Weissman, 2004). Adult stem cells are presumed quiescent within adult tissues, but divide infrequently to generate a stem cell clone and a transiently-amplifying cell. The transiently-amplifying cells will undergo a lim‐ ited number of cell divisions before terminal differentiation into mature functional tissue cells. The existence of adult stem cells has been reported in multiple organs; these include: brain, heart, skin, intestine, testis, muscle and blood, among others. This chapter focuses on four adult stem cell populations: hematopoietic, mesenchymal, periodontal ligament-de‐ rived, and spermatogonial (Table 1).


Introduction
Stem cells are undifferentiated cells defined by their abilities to self-renew and differentiate into mature cells. Stem cells found in fully developed tissues are defined as adult stem cells. The function of adult stem cells is the maintenance of adult tissue specificity by homeostatic cell replacement and tissue regeneration (Wagers and Weissman, 2004). Adult stem cells are presumed quiescent within adult tissues, but divide infrequently to generate a stem cell clone and a transiently-amplifying cell. The transiently-amplifying cells will undergo a limited number of cell divisions before terminal differentiation into mature functional tissue cells. The existence of adult stem cells has been reported in multiple organs; these include: brain, heart, skin, intestine, testis, muscle and blood, among others. This chapter focuses on four adult stem cell populations: hematopoietic, mesenchymal, periodontal ligament-derived, and spermatogonial (Table 1).
Hematopoietic stem cells are the most characterized adult stem cell population. They function to generate all cell lineages found in mature blood (erythroid, myeloid and lymphoid) and to sustain blood production during the entire life of an animal (Kondo et al., 2003). Adult bone marrow, umbilical cord blood and mobilized peripheral blood are sources of hematopoietic stem cells for transplantation in many blood-related diseases. Hematopoietic stem cells can be characterized by positive selection of CD34, CD45, and CD133 markers and negative selection of CD31, CD105 and CD146 markers (Tárnok et al., 2010).
Mesenchymal stem cells, also called marrow stromal cells, are another well-studied adult stem cell population. Mesenchymal stem cells were originally identified in the bone marrow, but have since been found in other systems such as adipose tissue, umbilical cord and menstrual blood (Ding et al., 2011). Mesenchymal stem cells differentiate into osteocytes, chondrocytes and adipocytes (Arita et al., 2011;Pittenger et al., 1999). Human mesenchymal stem cells can be characterized by the positive expression of CD29, CD44, CD73, CD90, CD105, CD146 and STRO-1, and the negative expression of CD31, CD34, CD45, CD49f and CD133 (Mödder et al., 2012;Tárnok et al., 2010).

Adult stem cells Feasible sources Characterization
Hematopoietic stem cells

Pluripotent stem cells
Pluripotency refers to the ability of cells to self-renew and differentiate into all 3 germ layers (ectoderm, endoderm and mesoderm). Pluripotent stem cells are the origin of all somatic and germ-line cells in the developing embryo. The first pluripotent cells were derived in 1976 from a type of germ-line tumor known as a teratocarcinoma (Hogan, 1976). Embryonic stem cells, derived from the inner cell mass of a blastocyst prior to gastrulation, are still considered the gold standard for pluripotent stem cells. Even though adult cells are terminally differentiated, pluripotency has also been conferred to these cells in past studies, by the technique of somatic cell nuclear transfer (Perry, 2005), parthenogenesis of unfertilized eggs (Brevini et al., 2008), and reprogramming by cell fusion (Pralong et al., 2006). Research into adult cell pluripotency was slow to progress un- Adult stem cells are thought to be tissue-specific and only able to differentiate into progeny cells of their tissues of origin. An increasing number of studies, however, report that adult stem cells are capable of giving rise to cells of an entirely distinct lineage. The concept of adult stem cell plasticity might be explained by 5 potential mechanisms: cell fusion, trans-differentiation, de-differentiation, heterogeneous stem cell populations, or pluripotency (Wagers and Weissman, 2004). Cell-cell fusion occurs at a low frequency, but is implicated in the transplantation of bone marrow cells to liver hepatocytes, cardiomyocytes and Purkinje neurons (Alvarez-Dolado et al., 2003). In cell fusion events, the stem cells acquire the mature phenotype of the tissue they are embedded within and can be easily mistaken for correct differentiation of the transplanted cells. Trans-differentiation is a direct lineage conversion by the activation of a dormant differentiation program to alter the lineage specificity of the cell. De-differentiation is another lineage conversion phenomenon in which a tissue-specific cell spontaneously de-differentiates into a more basal multipotent cell and re-differentiates to a new lineage. While the heterogeneity of the stem cell population employed can account for some of the apparent trans-differentiation and de-differentiation events observed in vivo, it is worth discussing as a separate factor in the resulting multi-lineage tissues, which are often seen after transplantation. The characterization of homogeneous stem cell populations that contribute to the regeneration of one cell type remains an active field of study for most cellular therapy applications. Lastly, pluripotent stem cells are present in adult tissues as minute sub-populations in certain stem cell niches. Such a population has already been identified and reported in bone marrow derived mesenchymal stem cells (Jiang et al., 2002). In addition, pluripotent stem cells in adult tissues can also arise from remnants of the migrating neural crest. The neural crest is a transient embryonic structure that affords various organs with cells which could undergo a more stochastic type of differentiation than other embryonic progenitor cells (Slack, 2008). Neural crest cells are pluripotent and may retain some of their characteristics after their migration and engraftment into their terminal sites.

Isolation of pluripotent adult stem cells
The expression of embryonic stem cell markers in some adult stem cells suggest a sub-population of pluripotent cells in these niches ( Table 2) (Conrad et al., 2008). Moreover, a human hematopoietic stem cell subpopulation, highly efficient in generating long-term multi-lineage grafts, can also be isolated by the same α 6 integrin expression (Notta et al., 2011). In addition, stem cells from granulocyte colony-stimulating factor-mobilized human peripheral blood can divide indefinitely without reaching replicative senescence and differentiate into multiple lineages (Cesselli et

Characterization of pluripotent adult stem cells
The standard tests for pluripotency are teratoma and chimera formation assays. Teratomas can be formed when pluripotent stem cells are injected into immunodeficient animals; they consist of foci with derivatives of ectodermal, mesodermal and endodermal embryonic germs layers . Chimeras can be generated when pluripotent stem cells are microinjected into mouse blastocysts and are induced to differentiate into multiple cell types during normal developmental processes (Becker et al., 1984). Teratoma formation assays can be used to test for the pluripotency of human stem cells, whereas both teratoma and chimera formation can test for the pluripotency of mouse stem cells. Spermatogonial stem cells isolated from human testis by positive expression of CD49f are able to form teratomas when injected into immunodeficient mice (Conrad et al., 2008). Mesenchymal stem cells isolated from murine bone marrow contribute to most of the somatic cell types (chimerism ranged between 0.1% and 45%) when they are singly injected into an early mouse blastocyst (Jiang et al., 2002). Moreover, human hem- Although most of the adult stem cells are unable to form teratomas in immunodeficient mice, can they still be defined as pluripotent stem cells? Considering this apparent inability as well as the variability in teratoma formation efficiency even when using a known pluripo-tent stem cell line, a teratoma assay might not be a suitable assay for pluripotency of adult stem cells. Instead, in vitro and in vivo differentiation into cells of the 3 embryonic germ layers along with chimera formation in xeno-transplanted mice can be applied for testing adult stem cell potency. The conventional concept of development involves a hierarchical structure of cellular commitment extending outward from embryonic and pluripotent, to adult terminally differentiated tissues. However, recent ideas propose that all or most tissues in the postnatal body are continuously turning over and contain a pluripotent stem cell reservoir (Slack, 2008). These pluripotent stem cell populations are able to differentiate into multiple cell types depending on their microenvironmental cues. Therefore, the stem cell status should be defined by plasticity (Zipori, 2005). . Therefore, these adult stem cells could also be defined as pluripotent stem cells.

Advantages of pluripotent adult stem cells over embryonic stem cells and induced pluripotent stem cells
Human embryonic stem cells come from the inner cell mass of human blastocysts. Therefore, embryonic stem cells used for cell therapy are allogenic; the transplanted donor cells do not originate from the recipient. This raises a concern about the immunogenic response of the host, and the need for immune-suppressive therapy concurrent with embryonic stem cell transplantation (Charron et al., 2009). Moreover, embryonic stem cellbased therapy has been hampered by the moral, legal and ethical dilemma surrounding the use of human embryos for derivation of the stem cell lines (Zarzeczny and Caulfield, 2009). Furthermore, as the gold standard of pluripotent stem cells, embryonic stem cells have the potential to form teratomas in the host. Tumorigenic potential can be reduced by differentiating the embryonic stem cells into lineage-specific progenitor cells or mature tissue cells prior to transplantation (Schwartz et al., 2012). In order to better control standards of good manufacturing practices and reduce variability as much as possible, the in vitro manipulation of embryonic stem cells should be minimized as recommend by the Food and Drug Administration (Lysaght and Campbell, 2011). Furthermore, tumorigenic potential remains a concern if the entirety of the embryonic stem cell population does not completely differentiate into fully mature cells.
Differentiated adult cells used for the generation of the induced pluripotent stem cells can be collected from the recipient body, avoiding the contentious need for a human embryo. This also circumvents the problem of immune rejection. There are technical hurdles, howev-er, concerning generation of induced pluripotent stem cells (Hayden, 2011). Firstly, the delivery of reprogramming factors (OCT4, SOX2, NANOG, LIN28, KLF4 and c-MYC) relies on the use of viral vectors for delivery (Takahashi et al., 2007). Retroviral sequences could integrate into the DNA of the host cells, potentially disrupting the gene structure as well as resulting in an aberrant phenotypic expression. Ultimately this could result in pathological mutations and cancer formation. Alternative methods such as direct protein or small molecule delivery have been adopted, although the reprogramming efficiency of these techniques is lower than with viral vectors (Kim et al., 2009;Shi et al., 2008). Secondly, two of the reprogramming factors, c-MYC and KLF4, are proto-oncogenes, which raise the concern of cancer formation further. The sources of adult stem cells are multiple and feasibly obtained from various adult tissues, such as bone marrow, blood, adipose tissue, teeth and testes (Table 1)

Potential applications of pluripotent adult stem cells
Stem cell clinical trials have advanced rapidly for a broad spectrum of diseases, such as diabetes, neurodegeneration, immune diseases, heart disease, and bone disease. In 2011, there were 123 clinical trials using mesenchymal stem cells (Trounson et al., 2011). It is predicted that stem cell therapy will eventually become the treatment of choice in regenerative medicine, especially the use of adult stem cells. As stem cell products become more wide-spread and maintained under various conditions, the need for global standardization and regulation of processes will become necessary for the viable application of these products in a clinical setting. The

Summary
Adult stem cells are found all over the body. They can be conveniently obtained from different accessible tissues: bone marrow, blood, adipose tissue, teeth and testes. Pluripotent adult stem cells, which reside as a subpopulation within adult stem cells, can be easily isolated by pluripotent cell surface markers, such as SSEA-3, SSEA-4 and CD49f. Moreover, pluripotent adult stem cells can be characterized by their ability to differentiate into cells of 3 germ layers (ectoderm, mesoderm and endoderm) as well as by the chimera formation in xeno-transplanted mice. Pluripotent adult stem cells are better than embryonic stem cells and induced pluripotent stem cells as they are an autologous source, require minimal manipulation and do not have the ability to form teratomas. In addition, they are more appropriate to be used as a clinical product for therapeutic treatments, as a cellular replacement or secretory protein reservoir. However, there are uncertainties that still remain unanswered. Which stem cell types are optimal for regenerative medicine? What is the optimal cell number for transplantation? Should the cells be preemptively differentiated or used as is? Further research is needed to understand the mechanisms of stem cells in regenerating damaged tissues after transplantation.