Progress in the reprogramming of somatic cells

Abstract

Pluripotent stem cells can differentiate into nearly all types of cells in the body. This unique potential provides significant promise for cell-based therapies to restore tissues or organs destroyed by injuries, degenerative diseases, aging, or cancer. The discovery of induced pluripotent stem cell (iPSC) technology offers a possible strategy to generate patient-specific pluripotent stem cells. However, because of concerns about the specificity, efficiency, kinetics, and safety of iPSC reprogramming, improvements or fundamental changes in this process are required before their effective clinical use. A chemical approach is regarded as a promising strategy to improve and change the iPSC process. Dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve iPSC reprogramming. In addition to the prospect of deriving patient-specific tissues and organs from iPSCs, another attractive strategy for regenerative medicine is transdifferentiation—the direct conversion of one somatic cell type to another. Recent studies revealed a new paradigm of transdifferentiation: using transcription factors (TFs) employed in iPSC generation to induce transdifferentiation, or iPSC TF-based transdifferentiation. This transdifferentiation not only reveals and utilizes the developmentally plastic intermediates generated during iPSC reprogramming, but also produces a very wide range of cells, including expandable tissue-specific precursor cells. Here, we review recent progress of small-molecule approaches in the generation of iPSCs. In addition, we summarize the new concept of iPSC TF–based transdifferentiation and discuss its application in generating various lineage-specific cells, especially cardiovascular cells.

Introduction

Pluripotent stem cells can self-renew indefinitely and undergo differentiation to produce all types of cells in adult bodies. They could be used to generate various cells or even tissues/organs for transplantation. Patients with injuries, degenerative disease, aging, or cancers would benefit from the realization of stem cell–based regenerative medicine. To avoid rejection by the patient’s immune system, cell-based therapies preferably use immune-matched donor cells, or the patient’s own cells, which could be derived from their own pluripotent stem cells.

Reprogramming to pluripotency

Various methods have been developed to reprogram cells to a pluripotent state. In the 1960s, somatic cell nuclear transfer (SCNT) was reported to erase lineage-specific signatures in the nuclei of a somatic cell and reprogram it to a totipotent state^{1, 2}. So far, SCNT approach has been successfully performed in mouse and some other species, but not yet reported in human. Fusion of somatic cells with pluripotent cells was another method proven to enable reprogramming somatic cells to pluripotent cells³, however the utility of this method is limited because the resultant cells are tetraploid. Both methods take advantage of cellular materials to establish pluripotency in somatic cells; SCNT uses material from oocytes, and cell fusion uses material from pluripotent cells. Although SCNT and cell fusion-induced reprogramming are determinative and relatively fast and efficient, significant technical challenges (as well as ethical challenges in the case of SCNT) remain before they can be practical. In addition, they entail complex mixtures of known and undefined factors from oocytes or pluripotent cells to trigger reprogramming, making mechanistic studies more challenging.

These barriers might be avoided by a new strategy in which mammalian somatic cells are reprogrammed to induced pluripotent stem cells (iPSCs) by ectopic expression of the pluripotent TFs Oct4, Sox2, Klf4, and c-Myc (or Nanog and Lin28 instead of Klf4 and c-Myc) ^4-6. In practice, combined with efficient differentiation strategies, iPSCs would be valuable not only to derive functional cells for transplantation, but also to establish patient-specific disease models for drug discovery and development. Similar to the capability of cell-type specific TFs to maintain cell identity by binding to specific DNA sequences across the genome and achieving additional sequence-binding specificity and transcriptional and epigenetic regulation by forming complexes with co-regulatory factors, exogenous iPSC TFs (TFs overexpressed in generation of iPSCs) cooperatively remodel chromatin to activate expression of genes in the pluripotency network and to suppress expression of genes that promote differentiation. Additionally, through both co-occupancy with downstream effectors of various signaling pathways and recruitment of diverse epigenetic enzymes over the whole genome, specific chromosomal binding patterns of exogenous iPSC TFs during the reprogramming process contribute to the establishment of iPSC-specific signal transduction, transcriptional circuitry, and epigenetic pattern. (Figure 1)

Figure 1. Mechanisms of reprogramming to pluripotency induced by exogenous transcription factors and hypothetical mechanisms of small molecule-mediated reprogramming to pluripotency.

A. Somatic cell maintains its identity by transcription factor (TF) mediated activation of lineage specific genes, and TF or heterochromatin mediated silencing of pluripoteny genes.

B. In the paradigm of TF-induced reprogramming to pluripotency, exogenous reprogramming TF complexes (e.g., pluripotency TF complexes coming from oocyte in SCNT, pluripotent cell in cell fusion, or ectopically expressed iPSC TFs), which can recognize and bind to specific sequences across the whole genome, interact with other TFs binding to nearby or distal sites and recruit other transcription cofactors (e.g., activators, repressors, and epigenetic enzyme complexes) to co-occupy and epigenetically modify the genome in a sequence-specific manner. As a result, the gene expression profile and epigenetic pattern of somatic cells gradually becomes iPSC specific. C. Unlike TFs, small molecules do not have the exquisite ability of molecular recognition possessed by TFs, and cannot specifically interact with both DNA and other transcription cofactors. Consequently, small molecules would hypothetically target endogenous components (e.g., TFs in purple and epigenetic enzyme complexes in light blue) in somatic cells to indirectly initiate iPSC reprogramming.

D. Pluripotent stem cell generated by either a TF or small-molecule approach maintains its pluripotency by TF-mediated activation of pluripoteny genes and repression of lineage specific genes.

Although iPSC reprogramming is technically simpler than SCNT and cell fusion, it only induces a stochastic and nonspecific reprogramming process and is therefore less efficient and slower than SCNT and cell fusion. This difference reflects the possibility that iPSC TFs are core components, but not the complete machinery functional in efficient and specific reprogramming induced by both SCNT and cell fusion. Besides, iPSCs generated by conventional methods raise concerns about their safety (e.g., immunogenicity⁷ and the risk of tumorigenesis) for clinical applications, as they employ virus-mediated gene delivery, which results in genomic integration of exogenous sequence, and enforced expression of oncogenes.

To increase efficiency, accelerate kinetics, and reduce safety concerns, many improvements in methodology have been achieved by different groups. Several specific cell types were shown to enable reprogramming with higher efficiency and/or less number of exogenous iPSC TFs⁸. However, nearly all of them rely on overexpression of exogenous iPSC TFs and extra manipulations (e.g., administration of cytokines or small molecules) to get reprogrammed efficiently and rapidly. In addition to reprogramming using different starting cell types, methods using virus-free^9-11, removable PiggyBac transposons¹², minicircle systems¹³ or episomal systems¹⁴ have been developed. Despite their success in generating iPSCs, often without a genetic footprint, use of DNA constructs leaves the possibility of genomic integration of exogenous sequence. Other attempts to generate iPSCs by nonintegrating virus-mediated gene delivery cannot preclude the safety concerns raised by using viruses^15-17. Although recombinant proteins or synthetic mRNAs can produce iPSCs, the protocols are costly and technically challenging^18-21. Recently, microRNA was used to generate reprogrammed iPSCs, but the practical utility and robustness of this approach are uncertain^{22, 23}.

Additionally, generation of iPSC with small molecules alone is being attempted. This promising strategy might eliminate many of the drawbacks (e.g., the risk of tumorigenesis from genomic integration of exogenous sequence or overexpression of oncogenes) of conventional and other improved iPSC reprogramming methods. Even if the final outcome of iPSC reprogramming induced by any method is the establishment of pluripotency associated gene expression profile and epigenetic pattern, the small molecule approach would employ a different process/mechanism from other methods to launch reprogramming process. In details, in other methods, exogenously introduced and pluripotency associated elements (e.g., TFs) trigger iPSC reprogramming by directly participating in and directing pluripotency-specific chromatin remodeling in somatic cells, whereas the small molecule approach indirectly initiates iPSC reprogramming by mediating endogenous, non-pluripotency-specific components in somatic cells (Figure 1). Therefore, at this point, successful induction of iPSCs by small molecules would fundamentally change the concept of iPSC reprogramming. Besides, all small molecules identified during development of this method would be possible candidates to further improve iPSC reprogramming and investigate the underlying mechanisms of this process. To date, many small molecules have been identified to modulate the induction of iPSCs, in both functionally replacing some exogenous iPSC TFs and significantly improving the efficiency and quality of iPSC reprogramming (Table 1).

Table 1.

Small molecules identified in iPSC research

Name & Structure	Target(s)/function	Effect(s)
BIX-01294	methyltransferase G9a inhibitor	Enables reprogramming of somatic cells by ectopic expression of Oct4 and Klf4 only.46,47
RG108	DNA methyltransferase inhibitor	In the presence of BIX-01294, improves reprogramming of MEFs induced by Oct4 and Klf4.46,47
Bayk8644 (BayK)	L-calcium channel agonist	In the presence of BIX-01294, improves reprogramming of MEFs induced by Oct4 and Klf4.46,47
Parnate	lysine-specific demethylase 1 inhibitor	In combination with CHIR99021, enables reprogramming of human keratinocytes induced by Oct4 and Klf4.50
5-Azacytidine (5-azaC)	DNA methyltransferase inhibitor	Improves MEF reprogramming efficiency with four iPSC TFs or under c-Myc-free condition.44,54 Facilitates erasure of epigenetic memory retained in established iPSCs.59
Suberoylanilide hydroxamic acid (SAHA)	HDAC inhibitor	Improves MEF reprogramming efficiency with four iPSC TFs.44,54
Trichostatin A (TSA)	HDAC inhibitor	Improves MEF reprogramming efficiency with four iPSC TFs.44,54 Facilitates erasure of epigenetic memory retained in established iPSCs.59
Valproic acid (VPA)	HDAC inhibitor	Improves MEF reprogramming efficiency with four iPSC TFs or under c-Myc-free condition.44,54 Enables reprogramming of human fibroblasts induced by Oct4 and Sox2.55
CHIR99021	Glycogen synthase kinase 3β inhibitor	Improves reprogramming efficiency of MEFs in the absence of Sox2 and cMyc.50 In combination with Parnate, enables reprogramming of human keratinocytes induced by Oct4 and Klf4.50
Kenpaullone	Glycogen synthase kinase 3β and cyclin-dependent kinases inhibitor	Functionally replaces Klf4 in reprogramming of MEFs in the presence of Oct4, Sox2, and cMyc.68
SB431542	TGFβ receptor inhibitor	Enhances MEF reprogramming efficiency in the absence of c-Myc.75, 76 Enhances and accelerates reprogramming of human somatic cells.69 Functionally replaces Sox2 in MEF reprogramming.75,76
PD0325901	MEK inhibitor	Enhances and accelerates reprogramming of somatic cells.69
Thiazovivin	ROCK inhibitor	Enhances and accelerates reprogramming of human somatic cells.69
A-83-01	TGFβ receptor inhibitor	Enhances MEF reprogramming.70-72 In combination with AMI-5, enables reprogramming of MEFs transduced with Oct4 only.73
AMI-5	Protein arginine methyltransferase inhibitor	In combination with A-83-01, enables reprogramming of MEFs transduced with Oct4 only.73
8-Bromoadenosine 3′, 5′-cyclic monophosphate (8-Br-cAMP)	Protein kinase A activator	Improves the reprogramming efficiency of human neonatal foreskin fibroblasts transduced with all four iPSC TFs.74
Dasatinib	Src family kinases Inhibitor	Functionally replaces Sox2 in MEF reprogramming.75,76
PP1	Src family kinases Inhibitor	Functionally replaces Sox2 in MEF reprogramming.75,76
iPYrazine (iPY)	Src family kinases Inhibitor	Functionally replaces Sox2 in MEF reprogramming.75,76
PS48	3′ phosphoinositide- dependent kinase 1 activator	In combination with A-83-01, PD0325901 and sodium butyrate, enables reprogramming of human somatic cells transduced with Oct4 only.92
Sodium butyrate	HDAC inhibitor	In combination with A-83-01, PD0325901, and PS48, enables reprogramming of human somatic cells transduced with Oct4 only.92
Vitamin C	Antioxidant and enzyme cofactor	Promotes iPSC generation from both mouse and human somatic cells.88
2, 4-Dinitrophenol	Oxidative phosphorylation un-coupler	Enhances efficiency of reprogramming to iPSC.92
Fructose 2, 6-bisphosphate	Phosphofructokinase 1 activator	Enhances efficiency of reprogramming to iPSC.92
N-oxalylglycine	Prolyl-4-hydroxylase inhibitor	Enhances efficiency of reprogramming to iPSC.92
Quercetin	Hypoxia-inducible factor pathway activator	Enhances efficiency of reprogramming to iPSC.92

Transdifferentiation

There are two promising strategies to regenerate tissue-specific cell types. In one, pluripotent cells are differentiated into various types of somatic cells. In the other, conventionally known as transdifferentiation, somatic cells are directly reprogrammed to another type of cells. Both approaches could be useful in the field of regenerative medicine and for disease modeling. Transdifferentiation has the advantage of avoiding the use of iPSCs to derive patient-specific cells, making the process faster and more efficient, reducing the risk of pluripotency associated tumorigenesis and probably avoiding immunogenicity identified recently in iPSCs⁷. In fact, many classic studies of transdifferentiation were thoroughly investigated before or around the time of discovery of iPSCs. For instance, ectopic expression of MyoD induced conversion of fibroblasts to myoblasts^{24, 25}, expression of GATA1 or PU1 promoted the reciprocal transition between myeloid cells and megakaryocyte/erythroid cells²⁶, expression of C/EBPα or C/EBPβ converted pre-B or pre-T cells to macrophages^{27, 28}, and expression of C/EBPβ provoked conversion from pancreatic cells to hepatocytes²⁹. However, most of these transitions were restricted to a relatively narrow lineage (e.g., hematopoietic, mesenchymal, or foregut endodermal system) and were induced by overexpression of a single TF. Success in generating iPSCs suggests constitutive expression of several TFs together might more efficiently induce transdifferentiation between less related cell types. This hypothesis has been verified. Many transdifferentiations induced by a set of lineage-specific TFs have been performed in vitro and in vivo^30-40. Additionally, a new concept, iPSC TF–based transdifferentiation, was recently proposed and demonstrated: transient overexpression of iPSC TFs in conjunction with cell-type-specific signals can reprogram somatic cells into diverse lineage-specific cells without going through the pluripotent state. Furthermore, compared with conventional transdifferentiation, this new method has many advantages, such as the use of universal TF system and the ability to generate a multipotent progenitor population.

Here, we will categorize all small molecules identified in iPSC research, based on their function in target cells, and review them separately. Then we will review iPSC TF-induced transdifferentiation and its use to transdifferentiate somatic cells into various linage-specific cells, especially cardiovascular cells.

1. Epigenetic modifiers

During reprogramming, cells not only undergo transcriptional changes but also exhibit epigenetic changes in DNA methylation and histone modifications^41-45. These changes convert the epigenetic pattern of somatic cells to an ESC-like state. Several small molecules that target enzymes involved in epigenetic modifications increase the efficiency of cellular reprogramming and sometimes can even functionally replace ectopic expression of certain TFs.

Dramatic changes in the histone methylation pattern are key features of iPSC reprogramming^{42, 43}. Thus, reprogramming would be affected by small molecules that target enzymes involved in histone methylation or demethylation. BIX-01294 (BIX), an inhibitor of histone H3K9 mono- and dimethyltransferase G9a, enables reprogramming by ectopic expression of Oct4 and Klf4 in somatic cells. Moreover, after treatment of neural progenitor cells (NPCs) with BIX-01294 or treatment of mouse embryonic fibrolasts (MEFs) with BIX-01294 combined with a DNA methyltransferase inhibitor RG108 or an L-calcium channel agonist Bayk8644 (BayK), reprogramming with only two TFs (i.e., Oct4 and Klf4) was as efficient as reprogramming with four TFs^{46, 47}. Consistently, G9a mediated H3K9 methylation is necessary for heterochromatinization and silencing of key pluripotency genes, such as Oct4 and Rex1, during early embryogenesis⁴⁸. Parnate is an inhibitor of lysine-specific demethylase 1 (LSD1) mediated H3K4 demethylation, shown to globally increase H3K4 methylation as well as transcriptional derepression of LSD1 target gene Oct4 in P19 embryonal carcinoma cells⁴⁹. In line with this observation, Parnate enabled two factors (Oct4 and Klf4) to induce conversion of human keratinocytes to iPSCs, when combined with CHIR99021 (an inhibitor of glycogen synthase kinase 3β )⁵⁰.

Because inhibition of histone deacetylation and DNA methylation improves the reprogramming efficiency of SCNT^51-53, it was hypothesized that such an approach might also aid in the establishment of iPSCs. As predicted, a DNA methyltransferase inhibitor, 5-azacytidine (5-azaC), or three histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA) and valproic acid (VPA) ), improves reprogramming efficiency after transduction of the four iPSC TFs in MEFs^{44, 54}. In addition, treatment with 5-azaC or VPA increases reprogramming efficiency under cMyc-free conditions^{44, 54}. Further mechanistic studies showed 5-azaC induced a rapid and stable transition of a fully reprogrammed iPSC state from a partially reprogrammed state with DNA hypermethylation at pluripotency genes and administration of VPA in MEFs upregulated some ESC-specific genes meanwhile downregulated some MEF-specific genes^{44, 54}. Moreover, VPA enables reprogramming of human fibroblasts transduced with only Oct4 and Sox2⁵⁵ and has been used to efficiently generate recombinant protein–induced pluripotent stem cells²¹.

Although mouse iPSC clones that are functionally identical to ESCs can be generated, subtle differences in gene expression and epigenetic patterns between iPSCs and ESCs could exist in some other iPSC clones⁴¹. Such differences could reflect residual expression and epigenetic pattern of original cell-type-specific genes, and/or gained genetic and epigenetic changes, contributed by incomplete epigenetic reprogramming or induced changes imposed by the reprogramming process^56-62. To improve upon such incomplete reprogramming, epigenetic memory can be largely erased by treating established iPSCs with 5-azaC and trichostatin A⁵⁹. On the other hand, such epigenetic memory may favor differentiation of iPSCs toward lineages related to its original cell type^{59, 61, 62}.

In summary, appropriate application of small molecules that function as epigenetic modifiers, either individually or in combination, significantly increases iPSC reprogramming efficiency. Furthermore, administration of epigenetic modifiers can largely diminish epigenetic memory retained in iPSCs, and thereby improve the quality of reprogrammed iPSCs.

2. Signaling modulators

Consistent with the notion that signal transduction pathways mediated by extrinsic factors and intrinsic transcriptional network cooperate to maintain self-renewal and pluripotency of ESCs, signal transduction pathways and TFs act coordinately to reprogram somatic cells to iPSCs⁶³. Therefore, small molecules that target signaling pathways would modulate reprogramming. Some such small molecules have been identified that increase reprogramming efficiency and can functionally replace some TFs in iPSC reprogramming.

Wnt signaling is important for maintaining the pluripotency of ESCs and for self-renewal of adult stem cells in multiple tissues⁶⁴. In ESCs, TCF3, a downstream effecter of the Wnt signaling pathway, co-occupies genome-wide loci with master pluripotency regulators, such as Oct4 and Nanog, and acts as a transcriptional repressor for targeted genes, competing activity of these master pluripotency regulators. β-Catenin, which is stabilized by activation of the Wnt signaling pathway, directly interacts with TCF3 and reduces inhibitory effect of TCF3 on pluripotency. Therefore, the Wnt signaling pathway is regarded as an integral component of the core transcriptional circuitry in ESCs⁶⁵. Indeed, β-catenin was involved in initial study of iPSC reprogramming and deletion of TCF3 could strikingly and rapidly enhance the efficiency of NPC reprogramming^{5, 66}. Consistently, Wnt3a enhanced the reprogramming of MEFs to pluripotency in the absence of cMyc⁶⁷; and CHIR99021, a glycogen synthase kinase 3β inhibitor that activates Wnt signaling, significantly improved reprogramming efficiency in MEFs in the absence of Sox2 and cMyc⁵⁰. In the same study, treatment with CHIR99021 and Parnate converted human keratinocytes to iPSCs upon ectopic expression of Oct4 and Klf4⁵⁰. Another glycogen synthase kinase 3β inhibitor, kenpaullone, which also inhibits other kinases, could replace Klf4 in reprogramming of MEFs in the presence of Oct4, Sox2, and cMyc. Interestingly, the role of kenpaullone in this process could not be replaced by CHIR99021, and the mechanism is unknown⁶⁸.

During reprogramming, mesenchymal-type fibroblasts undergo dramatic epithelial-like morphological changes and correlated gene expression changes, such as upregulation of E-cadherin (which is highly expressed in pluripotent cells) and concomitant downregulation of Snail. This so-called mesenchymal-to epithelial-transition (MET) inevitably occurs during successful reprogramming of cells. Therefore, small molecules that facilitate the MET process were hypothesized to enhance reprogramming⁶⁹. Indeed, small molecules that target three known MET mechanisms (for derepression of epithelial phenotype with upregulation and stabilization of E-cadherin), including inhibition of TGFβ receptor (by SB431542), MEK (by PD0325901), or ROCK (by thiazovivin), individually or in combination significantly enhanced reprogramming of human somatic cells and increased reprogramming speed⁶⁹. This MET mechanism was further characterized in three subsequent studies, where additional small molecules that inhibit the TGFβ pathway or upregulate E-cadherin were used^70-72. More recently, another TGFβ receptor inhibitor, A-83-01, combined with a protein arginine methyltransferase inhibitor, AMI-5, enabled reprogramming of MEFs transduced with Oct4 alone⁷³.

A few other small molecules that affect many other signaling pathways also facilitate iPSC reprogramming. A cyclic AMP analog, 8-bromoadenosine 3′, 5′-cyclic monophosphate (8-Br-cAMP), improved the reprogramming efficiency of human neonatal foreskin fibroblasts transduced with all four iPSC TFs⁷⁴. Inhibitors of Src family kinases, including Dasatinib, PP1, and iPYrazine (iPY), sufficiently supported reprogramming of MEFs in the absence of exogenous Sox2, as efficiently as TGFβ inhibitors^{75, 76}. All these observations are consistent with previously reports in which the PKA pathway and Src family kinases were shown to influence ESC self-renewal and/or differentiation^77-79.

In summary, small-molecule modulators of signaling pathways, individually or in combination, and sometimes even together with epigenetic modifiers, induce reprogramming with higher efficiency and/or fewer exogenous TFs, by affecting the integrated cellular network consisting of both signaling pathways and transcriptional circuitry.

3. Cell senescence alleviators

Cell senescence is typically seen during cellular reprogramming and is considered one of the barriers of reprogramming that account for the slow kinetics and low efficiency of this process⁸⁰. Indeed, the early stage of iPSC reprogramming entails a stress response with characteristics of cell senescence, including upregulated expression of p53, p21^CIP1, and p16^Ink4a/p19^{Arf 81}. Furthermore, downregulating the expression of any of these genes increased the efficiency and speed of iPSC reprogramming^82-87. A natural antioxidant, vitamin C, promoted iPSC generation from both mouse and human somatic cells through indirect reduction of p53 and p21^CIP1 expression and partial alleviation of cell senescence⁸⁸.

4. Metabolism regulators

Differentiated adult somatic cells typically use mitochondrial oxidative phosphorylation for cell growth, whereas pluripotent stem cells mainly use glycolytic metabolism^89-91. In rapidly proliferating pluripotent stem cells, glycolytic metabolism more effectively produces various macromolecular precursors to meet the metabolic/energy demand and generates fewer reactive oxygen species, which induce oxidative stress^{89, 91}. Therefore, transition from mitochondrial oxidative phosphorylation to glycolytic metabolism would be a crucial step in iPSC reprogramming^{91, 92}. This metabolic reprogramming is further supported by the ability of hypoxia-inducible factor 1α and cMyc to promote glycolytic metabolism and to improve the efficiency of iPSC reprogramming^{90, 93}. Consistently, a recent study showed PS48, a small-molecule activator of 3′ phosphoinositide-dependent kinase 1, combined with A-83-01, PD0325901, and sodium butyrate (a HDAC inhibitor), enabled reprogramming of human adult keratinocytes, umbilical vein endothelial cells, or amniotic fluid derived cells transduced with only Oct4, in which PS48 activated phosphatidylinositol 3-kinase (PI3K)/Akt pathway was shown to upregulate expression of several key glycolytic genes and facilitate the conversion from mitochondrial oxidative phosphorylation to glycolytic metabolism⁹². Remarkably, this study also revealed iPSC reprogramming efficiency was significantly increased by several other small molecules that enhance glycolytic metabolism and/or depress mitochondrial oxidative phosphorylation, including 2, 4-dinitrophenol (an oxidative phosphorylation uncoupler), fructose 2, 6-bisphosphate (an activator of phosphofructokinase 1), N-oxalylglycine (an inhibitor of prolyl-4-hydroxylase), and quercetin (an activator of hypoxia-inducible factor pathway)⁹². In a word, all evidences associated with these small molecules confirm the notion of metabolic reprogramming.

5. Trandifferentiation

Built upon iPSC reprogramming strategy and previous studies on cellular transdifferentiation restricted within several lineages, functional neurons, cardiomyocytes, hepatocytes, or macrophage-like cells can be converted from fibroblasts by ectopic expression of multiple lineage-specific TFs or microRNAs^30-38. Furthermore, transdifferentiation has also been induced with lineage-specific TFs in vivo^{39, 40}. However, transdifferentiated cells generated by ectopic expression of lineage-specific TFs were mostly one type of terminally differentiated cells. Therefore, conventional transdifferentiation typically would not allow isolation, expansion, and characterization of the reprogrammed cells, all of which are prerequisite for clinical application. In a word, the restricted proliferative capacity and limited cell type diversity of these transdifferentiated cells may substantially compromise their potential application in regenerative therapy.

Recently, a new paradigm of transdifferentiation was devised to generate various lineage-specific precursor cells by combining transient overexpression of the iPSC TFs and treatment with soluble signaling molecules. The basis of this universal transdifferentiation strategy is that conventional iPSC reprogramming proceeds as a slow, step-wise, and nondeterminative process that gives rise to iPSCs only at the late stage and with low efficiency and to populations of epigenetically unstable/plastic cells at early and intermediate stages. In addition, generating iPSCs requires an extended period (e.g., 8–12 days) of iPSC TF expression and a specific signaling environment (e.g., leukemia inhibitory factor). These observations suggest that temporal iPSC TF expression combined with different signaling environments (e.g., growth factors/cytokines, and small-molecule modulators of signaling pathways) would dictate reprogrammed cell fate. This would be consistent with the notion that cell-type-specific TFs direct different cell fates cooperatively with interacted signaling downstream TFs. Indeed, this concept and strategy was demonstrated by direct conversion of fibroblasts to cardiac⁹⁴, neural⁹⁵, or definitive endoderm (unpublished data) precursor cells using transient expression of iPSC TFs (e.g., 4 days), followed by treatment with BMP4, FGF4, or Activin-A, respectively. (Figure 2) Compared with conventional transdifferentiation, in which different cell specifications are determined by ectopic expression of different sets of lineage specific TFs, all iPSC TF-based transdifferentiations share the same TFs in the initial step: transient overexpression of iPSC TFs. Such transient gene expression might be more easily replaced with safer and more convenient methods without genetic modifications. Next, we will separately review iPSC TF–induced trandifferentiation toward different lineages.

Figure 2. A simplified and conceptual paradigm of iPSC TF–based transdifferentiation.

Temporally restricted overexpression of iPSC TFs in fibroblasts leads to the rapid generation of epigenetically “activated” cells, which can be further reprogrammed to iPSC by both extended expression of iPSC TFs and culture of iPSC medium, and parallelly can be coaxed by other signals and small molecule inhibitor of pluripotency to “relax” back into various differentiated state(s), ultimately giving rise to somatic cells entirely distinct from the starting population.

5.1 iPSC TF–based trandifferentiation of fibroblasts into cardiac cells

The mammalian heart lacks significant regenerative capacity, so transplantation of autologous cardiac cells generated in vitro is considered a possible therapy for heart disease. In a previous report, three cardiac TFs, Gata4, Mef2c, and Tbx5, enabled transdifferentiation of mouse postnatal cardiac or dermal fibroblasts directly into cardiomyocyte-like cells³¹. However, it took several weeks to generate beating cardiomyocytes, and the efficiency was very low. As an alternative method, temporal expression of iPSC TFs (Oct4/Sox2/Klf4/cMyc) in mouse fibroblasts for only 4–6 days with a JAK inhibitor and without LIF, followed by treatment with BMP4 in a chemically defined medium for additional 5 days, resulted in a large number of spontaneously contracting cardiomyocytes⁹⁴. This process was further characterized as a direct transdifferentiation, during which no pluripotent intermediates arose. Furthermore, an inverse relationship was demonstrated between induction of cardiomyocytes and generation of iPSCs, which could explain why cardiogenic transdifferentiation was facilitated by small-molecule inhibition of pluripotency but was impaired by prolonged overexpression of iPSC TFs⁹⁴.

Compared with the cardiac TF induced transdifferentiation, cardiogenic transdifferentiation triggered by transient expression of iPSC TFs followed by treatment with BMP4 is much faster (first spontaneous contractions beginning after 11 days versus 4–5 weeks) and more efficient.^{31, 94} Moreover, multipotent cardiovascular precursor cells arose at an intermediate stage, as suggested by the pattern of gene expression during iPSC TF induced transdifferentiation⁹⁴. If such cells could be isolated and expanded in vitro, they would eventually be a renewable source for unlimited amounts of cardiomyocytes and other terminally differentiated cardiovascular cells as well. Therefore, relative to cardiac TF induced terminally differentiated cardiomyocytes, which are restricted in cell type and limited in capacity to propagate after transplantation in vivo³¹, these multipotent cardiovascular precursor cells may be a more versatile cell source for modeling cardiovascular disease and for cell-based therapy.

For ultimate clinical applications, it would be highly desirable to develop a more robust condition for human system without any genetic modifications and safety concerns. Knowledge and techniques gained from development of iPSC reprogramming might be especially helpful to improve this new method of cardiogenic transdifferentiation.

5.2 iPSC TF–based trandifferentiation of fibroblasts toward other lineages

In addition to cardiac cells, other lineage-specific precursor cells, such as neural and definitive endoderm precursor cells, have also been generated from mouse and human fibroblasts by the same paradigm with different signaling molecules⁹⁵. In these studies, transient expression (4–6 days) of iPSC TFs in fibroblasts was the initial step shared by all. Another shared step was treatment with a JAK inhibitor to suppress the LIF-STAT3 pathway during iPSC reprogramming, which would prevent establishment of pluripotency in reprogrammed cells and facilitate the generation of developmentally plastic intermediate cells. The epigenetically activated cells were treated with FGF4 to generate neural precursor cells or with Activin-A to generate definitive endoderm precursor cells. Importantly, the efficiently and fast converted neural precursor cells could be expanded for serial passages and then differentiated into mature and subtype-specific neuronal cells and glial cells⁹⁵. Additionally, human fibroblasts have been directly converted to multipotent blood progenitors by prolonged ectopic expression of Oct4 and treatment with IGFII, bFGF, Flt3L and SCF⁹⁶.

In summary, cardiovascular cells and other lineage-specific precursor cells can be directly converted from iPSC TF-induced transdifferentiation, which implies that such strategy would provide a general platform to induce a broad range of cells for various applications.

Perspectives

The stem cell field has embarked on exciting discoveries that both iPSCs and lineage specific cells can be reprogrammed from somatic cells by ectopic expression of iPSC TFs. Because of the close relationship between these two types of reprogramming, they share some technical challenges and safety considerations that need to be addressed before their clinical applications. To date, several strategies have overcome these hurdles in respect of iPSC reprogramming, and some may also work well in iPSC TF–based transdifferentiation. As reviewed above, regarding iPSC reprogramming, small molecules are not only valuable to significantly promote cellular reprogramming and functionally substitute ectopic expression of TFs, but also provide insights into molecular mechanism underlying this process. Even though pleiotropy of small molecules may result in some side effects compromising the desired reprogramming process, appropriate employment of small molecule combinations would largely diminish these side effects and potentially have synergistic effects on reprogramming. In the future, complete small-molecule-based reprogramming will fundamentally change the reprogramming paradigm through a mechanism that involves activation of endogenous TFs by small molecules rather than by exogenously provided reprogramming TFs. Moreover, the success of small-molecule based transdifferentiation would greatly reduce safety concerns in the clinical application of reprogrammed cells by avoiding issues caused both by the generation of pluripotent cells (e.g., the risk of pluripotency associated tumorigenesis) and the introduction of exogenous TFs. Therefore, better understanding of these reprogramming processes and further development of these small molecules may ultimately be useful for in vivo stem cell biology and therapy.

Nonstandard abbreviations

iPSC

induced pluripotent stem cell

ESC

embryonic stem cell

NPC

neural progenitor cell

MEF

mouse embryonic fibroblast

TF

transcription factor

SCNT

somatic cell nuclear transfer

MET

mesenchymal-to-epithelial-transition

LSD1

lysine-specific demethylase 1

PI3K

phosphatidylinositol 3-kinase

BIX

BIX-01294

BayK

Bayk8644

5-azaC

5-azacytidine

HDAC

histone deacetylase

SAHA

suberoylanilide hydroxamic acid

TSA

trichostatin A

VPA

valproic acid

8-Br-cAMP

8-bromoadenosine 3′, 5′-cyclic monophosphate

iPY

iPYrazine