Toward Transgene-Free Induced Pluripotent Stem Cells: Lessons from Transdifferentiation Studies

Abstract

Regenerative medicine has received much attention over the years due to its clinical and commercial potential. The excitement around regenerative medicine waxes and wanes as new discoveries add to its foundation but are not immediately clinically applicable. The recent discovery of induced pluripotent stem cells has lead to a sustained effort from many research groups to develop clinically relevant regenerative medicine therapies. A major focus of cellular reprogramming is to generate safe cellular products through the use of proteins or small molecules instead of transgenes. The successful reprogramming of somatic nuclei to generate pluripotential cells capable of embryo development was pioneered over 50 years ago by Briggs and King and followed by Gurdon in the early 1960s. The success of these studies, the cloning of Dolly, and more current studies involving adult stem cells and transdifferentiation provide us with a large repository of potential candidate molecules and experimental systems that will assist in the generation of safe, transgene-free pluripotential cells.

What Can Transdifferentiation and Nuclear Reprogramming Teach Us about iPS Cell Generation

Despite the large amount of literature demonstrating transdifferentiation and cellular reprogramming, the ability of a differentiated cell to give rise to an unrelated cell type has been controversial. Prior to the induced pluripotent stem (iPS) cell studies, the idea of transdifferentiation or reprogramming was considered a rare event at best or a tissue culture artifact (Murry et al., 2004; Wagers et al., 2002). Many of the early studies reinforced this doubt, as many of the results were over interpreted. These studies relied on surface marker analysis of in vitro cultured cells to conclude a reprogramming event had occurred. Even if the studies contained in vivo results, usually no clear functional data was presented (Gang et al., 2004; Jiang et al., 2003; Rogers et al., 2007; Sanchez-Ramos et al., 2000; Schwartz et al., 2002; Wagers et al., 2002). A more stringent criteria for reprogramming was suggested that resulted in the inclusion of functional studies using animal models (Ellis et al., 2009; Maherali and Hochedlinger, 2008; Wagers et al., 2002). Implementation of these new criteria can be observed in many of the iPS cell reports. iPS cell studies have convinced the scientific community that differentiated human cells can be reprogrammed despite decades of dogma suggesting otherwise, and iPS cells have contributed to our understanding of these early reports on putative cell reprogramming using cell extracts or growth factors. Interestingly, many in the iPS field are now looking for nongenetic methods to reprogram cells using small molecules and growth factors. Recent studies using these nongenetic methods have been successful at generating both fully reprogrammed pluripotent cells and “partially” reprogrammed multipotential cells (Cho et al., 2010; Lin et al., 2009; Shi et al., 2008). Taken together, this suggests that some of the early (pre-iPS cell) reports regarding cellular transdifferentiation may be similar to the partially reprogrammed iPS colonies observed during cell reprogramming using the Yamanka factors, conferring merit to these studies (Mikkelsen et al., 2008). In this review we discuss the merits and the controversies surrounding those early reports and demonstrate that within these reports there may be some of the answers to nongenetic iPS reprogramming.

Reprogramming

King and Briggs (1957) carried out a number of studies demonstrating the progressive restriction of the nuclei as development proceeds. Although they did not observe normal development with nuclei from differentiated cells, many of the transferred nuclei were able to support limited development including gastrulation, suggesting their isolation from endoderm did not restrict them to this fate. Despite these results, later studies by Gurdon demonstrated that the cytoplasm of an enucleated oocyte is capable of reprogramming a differentiated nucleus. The first experiment to demonstrate nuclear reprogramming was the transfer of nuclei from differentiated Xenopus cells into enucleated oocytes resulted in normal feeding tadpoles. About 1.5% of transferred nuclei resulted in normal development. Gurdon also determined that the serially transfer of nuclei increased their developmental capacity, with a further 5.5% of the nuclei giving rise to feeding tadpoles. These studies demonstrate reprogramming of the nuclei by cytoplasmic factors. These early studies were important in that they demonstrated differentiation was reversible, and therefore did not involve changes in gene content or a permanent deletion of specific genes, but only a change in gene expression (Gurdon, 1962). Therefore, the underlying potential of a specified cell to differentiate into any cell type is retained. Understanding the maintenance of gene regulation will unlock nongenetic methods to reprogramming.

Tissue-Specific Stem Cells

There have been numerous reports of cells found in the skin (Toma et al., 2001), blood (Rogers et al., 2007), dental pulp (d'Aquino et al., 2009), and bone marrow (BM) (Jiang et al., 2002), that are capable of differentiating into multiple cell types. Whether this ability is due to tissue culture induced reprogramming, true transdifferentiation, or the presence of embryonic-like cells has not been determined in most cases. Our laboratory has focused on the ability of CD45⁺ blood cells to develop pluripotential properties only after cell culture in fibroblast growth factor 4 (FGF4), stem cell factor (SCF) and Fms-like tyrosine kinase receptor-3 ligand (Flt-3l). We have demonstrated that cell culture is a prerequisite for the appearance of blood cells that are capable of differentiating into cells representing all three germ layers (Rogers et al., 2007). We further argue that all other reports of multipotential or pluripotential cells from BM or umbilical cord blood could be explained the same way as isolation and characterization of the target cells always includes a time in culture for the cells.

An alternate explanation to culture induced reprogramming is that all adult stem cells are of a neural crest cell origin. Multipotent precursor cells reside in the skin (Toma et al., 2001) that share characteristics with embryonic neural crest stem cells (Fernandes et al., 2008). These skin-derived precursor cells (SKP) can differentiate into Schwann cells and myelinate axons in vitro and in vivo (McKenzie et al., 2006). Transplanted Schwann cells derived from the skin have been shown to myelinate host axons and result in neurological functional recovery in an experimental model of SCI (spinal cord injury) (Biernaskie et al., 2007). Furthermore, these cells can also differentiate into cells of endoderm and mesoderm origin.

Cell mixing is also a possible explanation because organs are composed of different cell types. For example, neurulation begins with ectodermal cells forming the neural plate along with mesodermal cells forming the notochord. At embryonic day 20 (human), the sides of the neural plate forms and somites derived from the mesoderm, which will eventually form the vertebrae, give structural support to the rising neural folds (Sadler, 2005). These neural folds fuse together forming the neural tube. While this is occurring, neural crest cells escape from the neural ectoderm and the neural epidermal junction and migrate into the mesoderm (Hall, 2008). After 28 days, the neural tube is closed, along with the posterior and anterior neuropores. The neural tube expands and the anterior end forms the brain, whereas the posterior end forms the spinal cord (Sadler, 2005). During these developmental events, there are opportunities for cells to intermix. Extensive migration of cells during development can also lead to cells of different germ cell origins being in the same location. Microglial cells are macrophages of the central nervous system (CNS) that migrate from the BM into the CNS early in development and therefore have a mesodermal origin (Ling and Wong, 1993). However, macroglial cells consist of astrocytes and oligodendrocytes and have the same ectodermal origin as neurons.

The issue, and confusion, regarding whether a stem cell resides in the tissues or is induced by culture, is best exemplified by mesenchymal stem cells. Mesenchymal stem cells (MSC) originate from the mesenchyme layer in the developing embryo, which eventually gives rise to muscles, blood, kidney, bone, and endothelial cells. MSC persist in tissues throughout the adult and can be isolated from BM, adipose tissue, muscle, peripheral blood, and to a certain extent, umbilical cord blood (Prockop, 1997; Smith et al., 2004; Wexler et al., 2003; Yu et al., 2004). Therefore, these cells can be harvested from these multiple sources, making them good candidates for autologous tissue transplantation. Studies have shown that these cells have a high capacity for self-renewal and are able to differentiate into various tissues.

Mesenchymal cells from the BM, which was the source of many transdifferentiation studies, are a naturally mixed population of fibroblast, osteoblast, muscle, and adipose cells, together making up a multipotential population. This does not mean there is a single multipotential stem cell. The isolation of a single clonal mesenchymal-derived cell that could give rise to the same range of cells argued for a multipotential MSC. The “problem” of whether or not this newly isolated cell was indeed a multipotential cell arose when neural cells were observed in the cultures (Chen et al., 2006; Fu et al., 2008; Jiang et al., 2003). Fibroblast, bone, adipocyte, and muscle are all mesoderm derived, whereas neural cells are ectoderm and therefore not of the same germ cell lineage. The presence of neural cells in the mix suggests a pluripotential population or a stem cell with properties similar to embryonic stem cells. Thus, clonality is important for distinguishing between the two. At issue is whether this mesoderm-based cell can produce neural cells that are of ectoderm origin or are the neural cells that arise contaminants, culture derived, or neural-like? Therefore, the shift from a mesoderm-derived mesenchymal cell to an ectoderm-derived neural cell represents a true reprogramming or transdifferentiation event, or the presence of an embryonic-like stem cell.

Other studies have also shown that mesenchymal cells can improve functional recovery when placed in an experimental model of SCI and differentiated into oligodendrocyte-like cells (Akiyama et al., 2002; Sasaki et al., 2001), suggesting transdifferentiation occurred. However, some reports contradict these results showing that no transdifferentiation occurred but functional improvement was still observed (Koda et al., 2005). It is unclear as to whether transplanted MSC cells demonstrating transdifferentiation fused with host neural cells making it only seem as if transdifferentiation occurred. A third possibility is that the time in culture prior to transplantation altered the developmental capacity of the cells. Several groups have differentiated MSC in culture and used nestin and glial fibrillary acidic protein (GFAP) as markers for neural differentiation (Phinney, 2007). However, these are not specific markers as these neural and oligodendrocyte markers are expressed in muscle and cartilage as well (Goolsby et al., 2003; Steidl et al., 2002). Electrophysiology studies or other functional tests were not used to demonstrate if these putative neural cells are functional so transdifferentiation cannot be confirmed. Although it would be ideal to have cells that meet the full pluripotential criteria, we need to keep our minds open to alternative hypothesis that may also prove valuable for clinical studies. Whether or not transplanted MSC are able to differentiate into neurons or oligodendrocytes, they have shown efficacy in functional recovery in experimental models of SCI, possibly through indirect mechanisms. MSC are able to modulate the immune system and reduce secondary injury (Aggarwal and Pittenger, 2005; Noel et al., 2007), secrete neurotrophic factors (Song et al., 2004) and increase vascularization and reduce cavity formation (Hofstetter et al., 2002). One study specifically used modified MSC to deliver neurotrophins to treat an experimental model of SCI and found that host axons were able to cross the glial scar, although no functional recovery was reported (Lu et al., 2007). Despite these uncertainties, clinical trials using MSC for SCI have already begun (Callera and do Nascimento, 2006; Yoon et al., 2007).

MSC have also been isolated from umbilical cord blood (UCB), although their isolation is rare and can only be obtained from fresh and not frozen UCB units within the first 5 h of collection (Mareschi et al., 2001; Wexler et al., 2003; Yu et al., 2004). UCB is an easily accessible source of cells, and therefore it holds an advantage as a source for MSC over other tissues. There have been a number of in vitro and in vivo studies demonstrating the capacity of UCB cells to differentiate into neural and glial cells and upon transplantation into animals have produced measurable functional improvements (Buzanska et al., 2002; Chen et al., 2005; Jang et al., 2004; Lee et al., 2007; Sanchez-Ramos et al., 2001). Although most studies that have focused on using UCB in regenerative medicine have specifically looked at a CD45 negative (CD45⁻) mesenchymal stem cell, our laboratory and others have demonstrated that CD45 positive (CD45⁺) blood cells can develop similar properties (Chua et al., 2010).

CD45⁺ Multipotential Stem Cell

The majority of multi- or pluripotential cells identified and reported from blood or BM sources are CD45 negative. This denotes that they are derived from the stromal or mesenchymal cells within the BM or UCB. These cells are usually considered to be mesenchymal progenitor cells or considered a MSC due to the population's ability to differentiate into a wider range of cells. Our laboratory has demonstrated that culturing CD45⁺/lineage negative (CD45⁺/Lin^neg) cells in serum-free culture supplemented with FGF4/SCF/Flt-3l, we could produce cells with an expanded differentiation potential (Chua et al., 2009, 2010; Rogers et al., 2007; Wong et al., 2010). These multipotential stem cells (MPSC) express Oct-4A and Nanog, the early tissue developmental markers nestin, desmin, GFAP, and Cfab1. After exposure to specialized differentiation media or in vivo methods we could demonstrate that CD45⁺/Lin^neg blood stem cells were capable of differentiating into osteoblasts, muscle, neural, islet precursors, endothelial cells, and hepatocytes. We have recently demonstrated that MPSC express Oct4A at levels equivalent to human embryonic stem (ES) cells and Oct4A is downregulated when the cells are induced to differentiate (Chua et al., 2009; Rogers et al., 2007). Yu et al. (2004), Krause et al. (2001), and Zhao et al. (2006) have also reported that multipotential CD45⁺ blood cells isolated from peripheral blood, UCB, and murine hematopoietic stem cells (HSC) after transplantation were capable of multilineage differentiation (Chua et al., 2010; Krause et al., 2001; Yu et al., 2004; Zhao et al., 2006).

Multipotent Adult Progenitor Cells

Although there has been much published data on the ability of tissue-specific stem cells to cross tissue boundaries, not much is known mechanistically about this process. The basic question of whether these cells represent a universal stem cell limited by its local tissue niche or whether these cells are capable of being reprogrammed during culture is unknown. Determining the basis of the pluripotent properties of these cells is further complicated by the fact that different cells have been identified. A good example of a universal stem cell is the BM MAPC (Jiang et al., 2002). MAPC have a wider differentiation potential than the normal mesenchymal progenitor. They are capable of differentiating into mesenchymal cell types, as well as cells of ectodermal origin such as neural cells. A cell similar to the MAPC has been reported for UCB (Kogler et al., 2004). Kogler et al. (2004) reported that 40% of cords could produce the universal somatic stem cell (USSC). Similar to the MAPCs, the USSCs also require culture in growth factor-enriched medium for their establishment, and have only been tested for their differentiation capacity after cell culture. USSCs are capable of differentiation into bone, cartilage, adipocyte, blood, neural, and liver cells. Although neural cell differentiation was interrogated in vivo, only one antibody was used (tau) and no functional studies or determination of cell fusion were carried out.

Putative pluripotent blood cells identified by Krause et al. (2001) were capable of multiorgan engraftment but contributed to a common cell type, the epithelium, of each of the organs, therefore demonstrating limited in vivo differentiation capability. In these experiments they started with long-term BM repopulating cells and found that these cells had the ability to change to cells of the epithelium. Another possibility is that although they used very few cells, it cannot be ruled out that their starting population was not completely void of mesenchymal cells.

Multipotential cells have also been observed in the fetal circulation between 7 and 12 weeks gestation (Campagnoli et al., 2001) as well as the human adult circulation (Kuznetsov et al., 2001). Although these cells express similar markers to that of BM mesenchymal cells, they have a greater developmental potential reminiscent of MAPCs and USSC. It is worth noting that the SKP cell, isolated from skin, has been attributed to a neural crest cell that remains from postembryonic stages (Fernandes et al., 2004). It is possible, although not yet proven, that MAPC and USSC and other tissue specific pluripotent stem cells are all neural crest derivatives.

Nuclear Reprogramming and Transdifferentiation: The Switch Between Neural Cells and Blood Cells

The term nuclear reprogramming is used to describe a differentiated cell that is induced to exhibit properties of an earlier developmental stage, thus gaining differentiation potential, which is then able to redifferentiate into another cell type. This can be done experimentally by somatic cell nuclear transfer (SCNT), cell fusion, or by the expression of certain transcription factors. An example of reprogramming is when a fibroblast cell is first induced to express Oct4 and Nanog (bringing the cell to a more primitive stage of development) and is then differentiated into a muscle cell and thus expresses MyoD followed by muscle actin and myosin heavy chain. Transdifferentiation or lineage conversion is a type of reprogramming where there is a direct change from one differentiated cell type to another (Hochedlinger and Plath, 2009). The conversion of blood to neural and the reverse can be used to illustrate the difficulties facing studies attempting to demonstrate transdifferentiation. The confounding results of the following studies and the ensuing debate illustrate some of the problems facing the field of transdifferentiation and argues that in vitro cell culture could account for some of the observations. Several groups had reported that neural stem cells (NSCs) can differentiate into blood cells (Bartlett, 1982; Bjornson et al., 1999; Clarke et al., 2000; Shih et al., 2001). Bjornson et al. (1999) reported that NSCs can differentiate into myeloid and lymphoid cells as well as early hematopoietic cells after injection of LacZ expressing NSC isolated from the brain or clonally derived NSC into sublethally irradiated mice resulted in LacZ-positive hematopoetic cells in the BM as assessed by colony forming unit assays and by flow cytometry. In a similar study by Shih et al. (2001), human fetal brain NSCs grown from neurospheres were used in long-term hematopoietic reconstitution of a severe combined immunodeficiency (SCID)-hu (severe combined immune deficient) mouse where 50% of the animals reconstituted with hematopoteic cells-derived from neurospheres. Conversely, another group repeated the experiments done by Bjornson et al. (1999) and found that they could not confirm that NSC are able to differentiate into hematopoetic cells (Morshead et al., 2002).

One possible reason for the opposing results is that the NSC's used were different in all cases. Others have criticized the Shih et al. (2001) article because it is not certain if the repopulating hematopoetic cells were indeed derived from NSC or from contaminating hematopoetic cells (Morshead et al., 2002). In the Morshead et al. (2002) article, the NSC used were passaged multiple times and therefore possibly transformed and were criticized for containing a low number of actual NSC (Shih et al., 2002). Bjornson et al. (1999) suggested that in the right environment transdifferentiation might be possible and that nuclear reprogramming is not necessarily required. The authors of the Morshead et al. (2002) article did observe that lengthy culture periods of NSC led to various changes in adhesion, growth characteristics, growth-factor dependence, and gene expression of the cells, suggesting that genetic or epigenetic changes could have resulted in the reported differentiation of NSC into HSC. In the end, all authors acknowledged that differentiation of NSC into hematopoetic cells is a rare event (Morshead et al., 2002; Shih et al., 2002; Vescovi et al., 2002).

Interestingly, it is now possible to generate iPS cells from various somatic cells, including mouse NSCs at high frequency (Kim et al., 2009). These neural-derived iPS cells are capable of differentiating into all three germ layers. Furthermore, recent studies have demonstrated that NSCs can be directly produced from fibroblasts without passing through an iPS cell intermediate by inducing the expression of three genes (Vierbuchen et al., 2010).

The studies demonstrating that blood can differentiate into neural cells and/or have a beneficial effect on animals suffering from SCI have revealed why we need to be careful when concluding that a transdifferentiation or reprogramming event has occurred. These studies also reveal alternative mechanisms of cell-based therapies. Although NSCs would be the obvious choice for treating SCI, as pointed out above, there are some hurdles to obtaining these cells for HLA-matched (human leukocyte antigen) transplantation (Eftekharpour et al., 2008; Morshead et al., 1998). Despite the obvious robust ability of NSC to replace damaged neurons or oligodendrocytes, alternative sources of stem cells are being sought. The studies using blood cells as a source of cells to treat SCI have yielded some very interesting results that highlight and explain some of the controversy surrounding the supposed properties of blood cells to differentiate into cells of neural lineages.

Although it has been shown that BM-derived HSC can differentiate into various cell types (Brazelton et al., 2000; Eglitis et al., 1999; Eglitis and Mezey, 1997; Krause et al., 2001; Lagasse et al., 2000), studies where BM cells were transplanted directly after isolation without any cell culture demonstrated that transdifferentiation does not occur or can be mistaken for other cellular events. For example, injection of HSCs into a growing tumor showed that the cells differentiated into endothelial-like cells that morphologically look like endothelial cells but did not express characteristic endothelial markers (Udani et al., 2005) and still expressed the pan-hematopoetic marker CD45. Other studies have found that fusion between transplanted cells and host cells can occur, which can be mistaken for transdifferentiation (Chung et al., 2002; Terada et al., 2002). We have demonstrated that the transplantation of cultured HSCs from human UCB resulted in a statistically significant improvement in SCI rats, but this was not due to donor cell engraftment and differentiation, but due to the factors secreted by the donor cells (Chua et al., 2010). Yet another possible explanation for transdifferentiation is the exchange of cell surface antigens, called trogocytosis (Yamanaka et al., 2009). The results of this study concluded human blood cells transplanted into mice formed hybrid blood cells containing both human and mouse antigens, which was not due to cell fusion.

Culture-Based Reprogramming, Are We There Yet?

We postulated that in vitro cell culture can lead to reprogramming, possibly to an embryonic-like stem cell or neural crest cell. The linear path taken during embryo development suggested that reprogramming would require extensive changes to the higher order chromatin. Data from experiments using cumulus cells as a nuclear donor in SCNT experiments suggest that reprogramming may not require that all higher order chromatin be reset. Despite the differentiated state of cumulus cells the Nanog and Sox2 promoters were undermethylated (Yamazaki et al., 2006). This suggests that some multipotent genes are primed for expression even in differentiated cells. The iPS experiments demonstrate that the transient expression of a few key genes is capable of initiating reprogramming. Therefore, specific cell culture conditions may override primary chromatin states resulting in reprogramming.

It has been demonstrated that coincubating Xenopus oocyte extracts with nonpermeabilized somatic cells is capable of causing reprogramming. This suggests that exogenous reprogramming factors can be introduced in standard tissue culture situations (Byrne et al., 2003; Miyamoto et al., 2007). Gurdon, in 1962, demonstrated that intestinal cell nuclei could support development to feeding tadpoles upon injection into enucleated oocytes. Later, he demonstrated that adult frogs could be produced the same way (Gurdon and Uehlinger, 1966), and eventually Gurdon proved that mouse fibroblast nuclei when injected into the germinal vesicle of Xenopus oocytes, could be reprogrammed to express Oct4 (Byrne et al., 2003). Similarly, Wilmut et al., in 1997, confirmed that embryo development is not irreversible by producing viable offspring from an adult somatic cell.

Cellular reprogramming of permeabilized fibroblasts was demonstrated by incubating them with ES cell extracts. The fibroblasts activated Oct 4, Sox2, c-myc, and Klf4 genes within 1–8 h. The fibroblasts continued to upregulate the pluripotency genes over the next 48 h (Bru et al., 2008). Recently, Cho et al. (2010) have demonstrated that permeabilized mouse fibroblasts coincubated with ES cell extracts are capable of being fully reprogrammed to iPS cells including being able to contribute to chimeras.

In order to illustrate that cell culture can induce Oct4 expression and reprogramming of differentiated cells into multipotential cells we cultured CD45⁺/ Lin^neg blood cells using FGF4 or FGF2, SCF and Flt-3l supplemented cultures we demonstrated that blood stem cells can be induced to express high levels of Oct4A, confirmed by PCR and immunocytochemistry (Wong et al., 2010). Clonal studies demonstrated that the Oct4+ cell is a product of the culture environment, and therefore must arise from the reprogramming of a blood cell. Furthermore, we were able to demonstrate that when Oct4+ cells were placed in neural differentiation medium, we could detect Oct4+/neurofilament+ transition cells. In addition, the cultured multipotential cells were CD45⁺ (a blood marker) and under differentiation conditions the cells could coexpress blood markers (CD45) and nonblood markers (CD31 for endothelial cells or neurofilament specific to neural cells). This coexpression was transient as the CD45 protein was downregulated as differentiation toward nonblood types progressed. We were also able to demonstrate epigenetic changes in the Oct4 promoter that correlates with the reactivation of the gene. These studies show that cell culture results in genomic alterations that lead to the generation of multipotential adult derived cells (Rogers et al., 2007; Wong et al., 2010). Furthermore, we were not able to successfully obtain neural cells directly from blood samples without first culturing them in expansion/reprogramming cultures, thus proving that initial cell culture is a mandatory step toward the development of multipotency of differentiated cells. Because the Lin^neg cells that we initially cultured were CD45⁺, they were not MSC. Furthermore, cell fusion and trogocytosis events can be ruled out because the differentiation of these cells and subsequent immunohistochemistry was done in vitro with no other cell types were present. Despite the claims in other studies that isolation and culture protocols are only expanding an already existing multipotential cell, in all cases the cell properties are only interrogated after culture (Durcova-Hills et al., 2006; Jiang et al., 2002; Kogler et al., 2004; McGuckin et al., 2008).

Durcova-Hill using a defined cell culture system, demonstrated that FGF2 was capable of initiating the reprogramming of primordial germ cells to pluripotent embryonic germ cells (Durcova-Hills et al., 2006). High concentrations of FGF2 were required but only for the first 24 h. After this point, the withdrawal of FGF2 did not affect the reprogramming process. Rather, endogenous FGF2 was upregulated, implying that it is important for maintenance of the pluripotent state. Furthermore, the cells required 9–10 days in culture to complete the reprogramming process, suggesting that FGF2 acts as an initiator that sets up a cascade of events that leads to a reprogrammed cell. The length of time required to accomplish reprogramming is analogous to the 12–16 days required for the formation of the blastema in newt appendage regeneration and the 20 days required for transformation of fibroblasts to iPS cells (Atkinson et al., 2006; Maherali, 2007; Tsonis and Del Rio-Tsonis, 2004). One mechanism that may explain the ability of FGF2 or FGF4 to initiate pluripotency is through the disruption of chromatin, specifically the polycomb group of genes. In zebrafish, blocking FGF receptors with SU5402 resulted in a change in the levels of expression of the polycomb gene ph2a (Komoike et al., 2005). This observation suggests that the polycomb complex may be the link between FGF signaling and the chromatin changes required for reprogramming. Therefore, a “general loosening” of the genome may result in the random activation of Oct4, Nanog, or Sox2, which will start a cascade of events leading to reprogramming. Although FGF2 and FGF4 have different roles in embryo development, they are capable of binding the same receptor and can be interchanged in the culture environment where receptor affinity is subjected to the concentration of the growth factor and the presence of heparin (Eswarakumar et al., 2005). FGF2 and FGF4 are both capable of maintaining the pluripotent state of embryos and human ES cells (Dvorak et al., 2005; Lamb and Rizzino, 1998; Vallier et al., 2005).

The laboratory of Sheng Ding demonstrated that sustained exposure of fibroblasts to the Oct4, Sox2, Klf4, and c-Myc proteins, could cause reprogramming in place of using transgenes (Zhou et al., 2009). This work opens up the possibility of nongenetic-based reprogramming. Other laboratories are now looking for replacement proteins that can mimic the effect of Oct4 or Sox2 but are either more stable or more potent. Furthermore, small molecule screens are producing positive hits for replacers of the Yamanaka factors (Li et al., 2011).

An important test for pluripotency and successful generation of iPS cells is the generation of teratomas. Therefore, the challenge is to generate safe iPS cells that have the potential to form teratomas, but at the same time, this property must be reversible in order to differentiate them down a defined pathway and produce cells or organs of a specific tissue type. Several ways to reduce the chances of teratoma formation include: (1) finding new approaches to generate iPS cells so that oncogenes are not permanently integrated into the genome or potential oncogenes inadvertently activated; (2) detect and remove undifferentiated iPS cells from differentiated tissues for transplantation, to eliminate the chances of teratomas arising from these cells. As mentioned above, some of the new approaches to generate iPS cells to make them safer include the use of proteins and small molecules, nonintegrating vectors and synthetic modified mRNA.

The experiments of Gurdon in the 1960's and the cloning of Dolly, among others, have clearly demonstrated that somatic cells can be reprogrammed using protein-based factors. Although the factors in these experiments remained undefined these experiments suggest that controlling reprogramming through in vitro tissue culture may be possible. Whether this will allow for the same level of control as the transgene experiments that have led to iPS cells needs to be investigated. The iPS experiments demonstrated that an exogenous initiation event is required but the newly activated endogenous genes can control the maintenance of pluripotency. This suggests that the right combination of growth factors may result in the initiation of reprogramming in a subset of cells and the newly reprogrammed cell will then maintain pluripotency (Bru et al., 2008; Miyamoto et al., 2007).

Footnotes

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

References

Aggarwal

, Pittenger

M.F.

2005. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 105:1815–1822.

Akiyama

, Radtke

, Kocsis

J.D.

2002. Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J. Neurosci., 22:6623–6630.

Atkinson

D.L.

, Stevenson

T.J.

, Park

E.J.

et al. 2006. Cellular electroporation induces dedifferentiation in intact newt limbs. Dev. Biol., 299:257–271.

Bartlett

P.F.

1982. Pluripotential hemopoietic stem cells in adult mouse brain. Proc. Natl. Acad. Sci. USA, 79:2722–2725.

Biernaskie

, Sparling

J.S.

, Liu

et al. 2007. Skin-derived precursors generate myelinating Schwann cells that promote remyelination and functional recovery after contusion spinal cord injury. J. Neurosci., 27:9545–9559.

Bjornson

C.R.

, Rietze

R.L.

, Reynolds

B.A.

et al. 1999. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science, 283:534–537.

Brazelton

T.R.

, Rossi

F.M.

, Keshet

G.I.

et al. 2000. From marrow to brain: expression of neuronal phenotypes in adult mice. Science, 290:1775–1779.

Bru

, Clarke

, McGrew

M.J.

et al. 2008. Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts. Exp. Cell Res., 314:2634–2642.

Buzanska

, Machaj

E.K.

, Zablocka

et al. 2002. Human cord blood-derived cells attain neuronal and glial features in vitro. J. Cell Sci., 115:2131–2138.

10.

Byrne

J.A.

, Simonsson

, Western

P.S.

et al. 2003. Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4 stem cell gene expression by amphibian oocytes. Curr. Biol., 13:1206–1213.

11.

Callera

, do Nascimento

R.X.

2006. Delivery of autologous bone marrow precursor cells into the spinal cord via lumbar puncture technique in patients with spinal cord injury: a preliminary safety study. Exp. Hematol., 34:130–131.

12.

Campagnoli

, Roberts

I.A.

, Kumar

et al. 2001. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood, 98:2396–2402.

13.

Chen

, Hudson

J.E.

, Walczak

et al. 2005. Human umbilical cord blood progenitors: the potential of these hematopoietic cells to become neural. Stem Cells, 23:1560–1570.

14.

Chen

, Teng

F.Y.

, Tang

B.L.

2006. Coaxing bone marrow stromal mesenchymal stem cells towards neuronal differentiation: progress and uncertainties. Cell Mol. Life Sci., 63:1649–1657.

15.

Cho

H.J.

, Lee

C.S.

, Kwon

Y.W.

et al. 2010. Induction of pluripotent stem cells from adult somatic cells by protein-based reprogramming without genetic manipulation. Blood, 116:386–395.

16.

Chua

S.J.

, Bielecki

, Wong

C.J.

et al. 2009. Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture. Biochem. Biophys. Res. Commun., 379:217–221.

17.

Chua

S.J.

, Bielecki

, Yamanaka

et al. 2010. The effect of umbilical cord blood cells on outcomes after experimental traumatic spinal cord injury. Spine (Phila Pa 1976), 35:1520–1526.

18.

Chung

Y.S.

, Zhang

W.J.

, Arentson

et al. 2002. Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression. Development, 129:5511–5520.

19.

Clarke

D.L.

, Johansson

C.B.

, Wilbertz

et al. 2000. Generalized potential of adult neural stem cells. Science, 288:1660–1663.

20.

d'Aquino

, De Rosa

, Laino

et al. 2009. Human dental pulp stem cells: from biology to clinical applications. J. Exp. Zool. B Mol. Dev. Evol., 312B:408–415.

21.

Durcova-Hills

, Adams

I.R.

, Barton

S.C.

et al. 2006. The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells. Stem Cells, 24:1441–1449.

22.

Dvorak

, Dvorakova

, Koskova

et al. 2005. Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cells, 23:1200–1211.

23.

Eftekharpour

, Karimi-Abdolrezaee

, Fehlings

M.G.

2008. Current status of experimental cell replacement approaches to spinal cord injury. Neurosurg. Focus, 24:E19.

24.

Eglitis

M.A.

, Mezey

1997. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc. Natl. Acad. Sci. USA, 94:4080–4085.

25.

Eglitis

M.A.

, Dawson

, Park

K.W.

et al. 1999. Targeting of marrow-derived astrocytes to the ischemic brain. Neuroreport, 10:1289–1292.

26.

Ellis

, Bruneau

B.G.

, Keller

et al. 2009. Alternative induced pluripotent stem cell characterization criteria for in vitro applications. Cell Stem Cell, 4:198–199author reply 202.

27.

Eswarakumar

V.P.

, Lax

, Schlessinger

2005. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev., 16:139–149.

28.

Fernandes

K.J.

, McKenzie

I.A.

, Mill

et al. 2004. A dermal niche for multipotent adult skin-derived precursor cells. Nat. Cell Biol., 6:1082–1093.

29.

Fernandes

K.J.

, Toma

J.G.

, Miller

F.D.

2008. Multipotent skin-derived precursors: adult neural crest-related precursors with therapeutic potential. Philos. Trans. R. Soc. Lond. B Biol. Sci., 363:185–198.

30.

, Zhu

, Huang

et al. 2008. Derivation of neural stem cells from mesenchymal stemcells: evidence for a bipotential stem cell population. Stem Cells Dev., 17:1109–1121.

31.

Gang

E.J.

, Jeong

J.A.

, Hong

S.H.

et al. 2004. Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilical cord blood. Stem Cells, 22:617–724.

32.

Goolsby

, Marty

M.C.

, Heletz

et al. 2003. Hematopoietic progenitors express neural genes. Proc. Natl. Acad. Sci. USA, 100:14926–14931.

33.

Gurdon

J.B.

1962. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J. Embryol. Exp. Morphol., 10:622–640.

34.

Gurdon

J.B.

, Uehlinger

1966. “Fertile” intestine nuclei. Nature, 210:1240–1241.

35.

Hall

B.K.

2008. The neural crest and neural crest cells: discovery and significance for theories of embryonic organization. J. Biosci., 33:781–793.

36.

Hochedlinger

, Plath

2009. Epigenetic reprogramming and induced pluripotency. Development, 136:509–523.

37.

Hofstetter

C.P.

, Schwarz

E.J.

, Hess

et al. 2002. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc. Natl. Acad. Sci. USA, 99:2199–2204.

38.

Jang

Y.K.

, Park

J.J.

, Lee

M.C.

et al. 2004. Retinoic acid-mediated induction of neurons and glial cells from human umbilical cord-derived hematopoietic stem cells. J. Neurosci. Res., 75:573–584.

39.

Jiang

, Jahagirdar

B.N.

, Reinhardt

R.L.

et al. 2002. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 418:41–49.

40.

Jiang

, Henderson

, Blackstad

et al. 2003. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc. Natl. Acad. Sci. USA, 100,Suppl. 1:11854–11860.

41.

Kim

J.B.

, Zaehres

, Arauzo-Bravo

M.J.

et al. 2009. Generation of induced pluripotent stem cells from neural stem cells. Nat. Protoc., 4:1464–1470.

42.

King

T.J.

, Briggs

1957. Changes in the nuclei of differentiating gastrula cells, as demonstrated by nuclear transplantation. Proc. Natl. Acad. Sci. USA, 41:321–325.

43.

Koda

, Okada

, Nakayama

et al. 2005. Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice. Neuroreport, 16:1763–1767.

44.

Kogler

, Sensken

, Airey

J.A.

et al. 2004. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J. Exp. Med., 200:123–135.

45.

Komoike

, Kawamura

, Shindo

et al. 2005. Zebrafish Polycomb group gene ph2alpha is required for epiboly and tailbud formation acting downstream of FGF signaling. Biochem. Biophys. Res. Commun., 328:858–866.

46.

Krause

D.S.

, Theise

N.D.

, Collector

M.I.

et al. 2001. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell, 105:369–377.

47.

Kuznetsov

S.A.

, Mankani

M.H.

, Gronthos

et al. 2001. Circulating skeletal stem cells. J. Cell Biol., 153:1133–1140.

48.

Lagasse

, Connors

, Al-Dhalimy

et al. 2000. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat. Med., 6:1229–1234.

49.

Lamb

K.A.

, Rizzino

1998. Effects of differentiation on the transcriptional regulation of the FGF-4 gene: critical roles played by a distal enhancer. Mol. Reprod. Dev., 51:218–224.

50.

Lee

M.W.

, Moon

Y.J.

, Yang

M.S.

et al. 2007. Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood. Biochem. Biophys. Res. Commun., 358:637–643.

51.

, Zhang

, Yin

et al. 2011. Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res., 21:196–204.

52.

Lin

, Ambasudhan

, Yuan

et al. 2009. A chemical platform for improved induction of human iPSCs. Nat. Methods, 6:805–808.

53.

Ling

E.A.

, Wong

W.C.

1993. The origin and nature of ramified and amoeboid microglia: a historical review and current concepts. Glia, 7:9–18.

54.

, Jones

L.L.

, Tuszynski

M.H.

2007. Axon regeneration through scars and into sites of chronic spinal cord injury. Exp. Neurol., 203:8–21.

55.

Maherali

2007. Directly reprogrammed fibroblasts show global Epigeneitc Remodeling and wide spread tissue contribution. Cell Stem Cell, 1:55–70.

56.

Maherali

, Hochedlinger

2008. Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell, 3:595–605.

57.

Mareschi

, Biasin

, Piacibello

et al. 2001. Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Haematologica, 86:1099–1100.

58.

McGuckin

, Jurga

, Ali

et al. 2008. Culture of embryonic-like stem cells from human umbilical cord blood and onward differentiation to neural cells in vitro. Nat. Protoc., 3:1046–1055.

59.

McKenzie

I.A.

, Biernaskie

, Toma

J.G.

et al. 2006. Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system. J. Neurosci., 26:6651–6660.

60.

Mikkelsen

T.S.

, Hanna

, Zhang

et al. 2008. Dissecting direct reprogramming through integrative genomic analysis. Nature, 454:49–55.

61.

Miyamoto

, Furusawa

, Ohnuki

et al. 2007. Reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts. Mol. Reprod. Dev., 74:1268–1277.

62.

Morshead

C.M.

, Craig

C.G.

, van der Kooy

1998. In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain. Development, 125:2251–2261.

63.

Morshead

C.M.

, Benveniste

, Iscove

N.N.

et al. 2002. Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations. Nat. Med., 8:268–273.

64.

Murry

C.E.

, Soonpaa

M.H.

, Reinecke

et al. 2004. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature, 428:664–668.

65.

Noel

, Djouad

, Bouffi

et al. 2007. Multipotent mesenchymal stromal cells and immune tolerance. Leuk. Lymphoma, 48:1283–1289.

66.

Phinney

D.G.

2007. Biochemical heterogeneity of mesenchymal stem cell populations: clues to their therapeutic efficacy. Cell Cycle, 6:2884–2889.

67.

Prockop

D.J.

1997. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 276:71–74.

68.

Rogers

, Yamanaka

, Bielecki

et al. 2007. Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation. Exp. Cell Res., 313:1839–1852.

69.

Sadler

T.W.

2005. Embryology of neural tube development. Am. J. Med. Genet. C Semin. Med. Genet., 135C:2–8.

70.

Sanchez-Ramos

, Song

, Cardozo-Pelaez

et al. 2000. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp. Neurol., 164:247–256.

71.

Sanchez-Ramos

J.R.

, Song

, Kamath

S.G.

et al. 2001. Expression of neural markers in human umbilical cord blood. Exp. Neurol., 171:109–115.

72.

Sasaki

, Honmou

, Akiyama

et al. 2001. Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons. Glia, 35:26–34.

73.

Schwartz

R.E.

, Reyes

, Koodie

et al. 2002. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J. Clin. Invest., 109:1291–1302.

74.

Shi

, Do

J.T.

, Desponts

et al. 2008. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell, 2:525–528.

75.

Shih

C.C.

, Weng

, Mamelak

et al. 2001. Identification of a candidate human neurohematopoietic stem-cell population. Blood, 98:2412–2422.

76.

Shih

C.C.

, Mamelak

, LeBon

et al. 2002. Hematopoietic potential of neural stem cells. Nat. Med., 8:535–536; author reply 536–537.

77.

Smith

J.R.

, Pochampally

, Perry

et al. 2004. Isolation of a highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma. Stem Cells, 22:823–831.

78.

Song

, Kamath

, Mosquera

et al. 2004. Expression of brain natriuretic peptide by human bone marrow stromal cells. Exp. Neurol., 185:191–197.

79.

Steidl

, Kronenwett

, Rohr

U.P.

et al. 2002. Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells. Blood, 99:2037–2044.

80.

Terada

, Hamazaki

, Oka

et al. 2002. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature, 416:542–525.

81.

Toma

J.G.

, Akhavan

, Fernandes

K.J.

et al. 2001. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat. Cell Biol., 3:778–784.

82.

Tsonis

P.A.

, Del Rio-Tsonis

2004. Lens and retina regeneration: transdifferentiation, stem cells and clinical applications. Exp. Eye Res., 78:161–72.

83.

Udani

V.M.

, Santarelli

J.G.

, Yung

Y.C.

et al. 2005. Hematopoietic stem cells give rise to perivascular endothelial-like cells during brain tumor angiogenesis. Stem Cells Dev, 14:478–486.

84.

Vallier

, Alexander

, Pedersen

R.A.

2005. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J. Cell Sci., 118:4495–4509.

85.

Vescovi

A.L.

, Rietze

, Magli

M.C.

et al. 2002. Hematopoietic potential of neural stem cells. Nat. Med., 8:535; author reply 536–537.

86.

Vierbuchen

, Ostermeier

, Pang

Z.P.

et al. 2010. Direct conversion of fibroblasts to functional neurons by defined factors. Nature, 463:1035–1041.

87.

Wagers

A.J.

, Sherwood

R.I.

, Christensen

J.L.

et al. 2002. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science, 297:2256–2269.

88.

Wexler

S.A.

, Donaldson

, Denning-Kendall

et al. 2003. Adult bone marrow is a rich source of human mesenchymal “stem” cells but umbilical cord and mobilized adult blood are not. Br. J. Haematol., 121:368–374.

89.

Wilmut

, Schnieke

A.E.

, McWhir

et al. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature, 385:810–813.

90.

Wong

C.J.

, Casper

R.F.

, Rogers

I.M.

2010. Epigenetic changes to human umbilical cord blood cells cultured with three proteins indicate partial reprogramming to a pluripotent state. Exp. Cell Res., 316:927–939.

91.

Yamanaka

, Wong

C.J.

, Gertsenstein

et al. 2009. Bone marrow transplantation results in human donor blood cells acquiring and displaying mouse recipient class I MHC and CD45 antigens on their surface. PLoS One, 4:e8489.

92.

Yamazaki

, Fujita

T.C.

, Low

E.W.

et al. 2006. Gradual DNA demethylation of the Oct4 promoter in cloned mouse embryos. Mol. Reprod. Dev., 73:180–188.

93.

Yoon

S.H.

, Shim

Y.S.

, Park

Y.H.

et al. 2007. Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: Phase I/II clinical trial. Stem Cells, 25:2066–2073.

94.

, Xiao

, Shen

et al. 2004. Mid-trimester fetal blood-derived adherent cells share characteristics similar to mesenchymal stem cells but full-term umbilical cord blood does not. Br. J. Haematol., 124:666–675.

95.

Zhao

, Wang

, Mazzone

2006. Identification of stem cells from human umbilical cord blood with embryonic and hematopoietic characteristics. Exp. Cell Res., 312:2454–2464.

96.

Zhou

, Wu

, Joo

J.Y.

et al. 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 4:381–384.

Toward Transgene-Free Induced Pluripotent Stem Cells: Lessons from Transdifferentiation Studies

Abstract

Abstract

What Can Transdifferentiation and Nuclear Reprogramming Teach Us about iPS Cell Generation

Reprogramming

Tissue-Specific Stem Cells

CD45+ Multipotential Stem Cell

Multipotent Adult Progenitor Cells

Nuclear Reprogramming and Transdifferentiation: The Switch Between Neural Cells and Blood Cells

Culture-Based Reprogramming, Are We There Yet?

Footnotes

Author Disclosure Statement

References

CD45⁺ Multipotential Stem Cell