Direct Reprogramming Facilitated by Small Molecules

The ability to reprogram cellular fate provide great hope for regenerative medicine and offer excellent cellular resources for disease modeling and drug discovery. Direct reprogramming, also called lineage reprogramming or transdifferentiation, is the process of changing one cell type to another without going through an intermediate pluripotent step. Direct reprogramming is generally achieved using lineage-specific transcription factors or microRNAs. The efficiency of direct reprogramming is usually relatively low without the aid of small molecule compounds. By using small molecules in conjunction with transcription factors or microRNAs, the efficiency of direct reprogramming is greatly increased and sometimes even less transgenes are required. Common targets of the small molecules used in direct reprogramming include epigenetic regulators, such as DNA methyltransferases and histone deacetylases, and signaling molecules, such as GSK3β and TGFβ pathways. By modulating epigenetic landscape and signaling pathways critical for cell fate determination, these small molecules allow cell fate conversion in a rapid and efficient manner. This review aims to highlight the versatile roles of small molecules in direct reprogramming.


Introduction
The ability to reprogram differentiated cells into a pluripotent state has revolutionized the field of regenerative medicine.The therapeutic potential of this process is vast and diverse, providing the hope to cure diseases and injuries that currently have no treatment or cure.However, because procedures for reprogramming into pluripotent stem cells often involve the use of oncogenes, problems such as tumorigenicity must be overcome before induced pluripotent stem cells (iPSCs) can be used in clinical settings [1].An alternative process, termed direct reprogramming, allows cell fate change without going through a pluripotent stage.The direct reprogramming process involves converting one fully or partially differentiated cell type into another fully or partially differentiated cell type through the use of lineage-specific transcription factors or micro RNAs.However, the efficiency of direct reprogramming without the aid of small molecules has been relatively low [2][3][4].Recently, the use of small molecules in facilitating this process has become more widespread.Small molecule-facilitated direct reprogramming often results in efficiencies much higher than that without the aid of small molecules [2][3][4][5][6][7][8][9].This review article will discuss the effect of various small molecules on direct reprogramming and the possible mechanisms of action for these small molecules (Table 1).

Small molecules facilitate direct reprogramming into neurons and neural stem cells
Direct reprogramming of fibroblasts and other somatic cell types into induced neurons and induced neural stem cells is one of the most heavily investigated topics within the direct reprogramming field.The potential therapeutic applications of the directly induced neural cells are numerous.The induced neurons or neural stem cells could be used to replace damaged nervous tissue or even regenerate this tissue in vivo.Multiple studies have used small molecule compounds to facilitate direct reprogramming and the impact of these small molecules in the reprogramming process is astounding.The following studies all used small molecules in direct reprogramming into neural cells and are discussed in chronological order.
The small molecules SB431542 (a TGFβ inhibitor) and CHIR99021 (a GSK3β inhibitor) were used to facilitate reprogramming human postnatal fibroblast cells into induced neurons in a study published in 2012 [3].These small molecules were chosen for their inhibitory effects on the SMAD and GSK3β signaling pathways, which is thought to increase the efficiency of reprogramming into neurons.After transducing human fibroblasts with the gene of Ascl1 (Achaete-scute homolog 1), a neurogenic transcription factor, and the gene encoding another transcription factor, such as Olig2, Brn2, Zic1, Myt1L or Ngn2, these cells could be converted into neurons, but with less than 5% conversion efficiency.Treatment of the transduced cells (e.g.Ascl1 and Ngn2transduced cells) with CHIR99021 alone, SB431542 together with Noggin (a protein inhibitor of bone morphogenetic protein [BMP] signaling), or all three molecules for 12 days had 4-fold, 10-fold, and 17-fold increase in conversion efficiency, respectively.The induced neuron-like cells expressed the neuronal marker βIII tubulin, and the neuronal subtype marker γ-aminobutyric acid (GABA) or vesicular glutamate transporter 1 (VGLUT1), and were able to generate action potentials and form synaptic networks, indicating their functionality.The same experiments were repeated with cord blood-derived stem cells (CBSCs) and produced similar results.Direct reprogramming achieved by combining two transgenes (Ascl1 and Ngn2), two small molecules (SB431542 and CHIR99021) and Noggin reached more than 100% conversion yield and more than 60% neuronal purity [3].
Direct reprogramming of human mesenchymal stem cells (MSCs) into neuron-like cells has been achieved by introducing dorsomorphin and SB431542 into a previously described neuronal induction cocktail containing epigenetic and cAMP modifiers, including Trichostatin A (TSA), RG-108, 8-BrcAMP, and Rolipram [10,11].Dorsomorphin and SB431542 were chosen for this study because of their inhibitory effects on SMAD signaling [10].Simultaneous inhibition of both the activin/nodal and BMP pathways is accomplished through the use of dorsomorphin and SB431542.When human MSCs were cultured in NeuroCult basal medium, supplemented with N2, basic FGF and the small molecule cocktail containing dorsomorphin and SB431542, dendrites and axon-like processes could be observed in 30-50% of the cells at 3-5 days after neural induction.After 2 to 3 weeks, about 95% of the cells expressed markers for neural progenitors and mature neurons.The induced neuron-like cells were also shown to form synapse-like structures when co-cultured with human neural stem cells [10].The MSC-derived neuron like cells induced by dorsomorphin and SB431542-containing small molecule cocktail also exhibited dopaminergic neuronal properties as revealed by an increase in dopamine release [12].
In a parallel study, the small molecules forskolin (FSK, a cAMP activator) and dorsomorphin increased the efficiency of direct reprogramming of human fetal lung fibroblasts into cholinergic neurons [13].Human fetal lung fibroblast cells were transduced with retrovirus expressing the neurogenic transcription factor neurogenin 2 (NGN2) and treated with one of seven different small molecules after viral transduction.Treating the NGN2-transduced fibroblasts with FSK induced βIII tubulin (Tuj1)-positive cells with neuron-like morphology.These Tuj1-positive cells became visible after 7 days post viral infection (d.p.i.), but could only survive to 10 d.p.i and died before maturation.When the NGN2transduced fibroblasts were treated with a combination of FSK and dorsomorphin, most transduced cells survived and expressed the mature neuronal marker MAP2 at 14 d.p.i.It is interesting to note that the effect of FSK and dorsomorphin on promoting transcription factor-induced reprogramming into neurons was unique to NGN2, because treating other transcription factortransduced fibroblasts with FSK and dorsomorphin under the same culture condition failed to induce robust neuronal reprogramming.The neurons induced by the small molecules and Ngn2 exhibited typical neuronal morphology and electrophysiological properties.Throughout the reprogramming process, Sox2, Olig2, and Pax6 were not detectable, indicating that the induced neurons did not undergo a progenitor state [13].
In another paper published in 2013, a single treatment with the cancer drug decitabine, or 5-aza-2'-deoxycytidine (a DNMT inhibitor), allowed direct reprogramming of human skin keratinocytes into neuron-like cells when cultured in neuronal induction media for 5 days, followed by neuronal maintenance media for another 7 days [14].Treatment with decitabine followed by culturing in neuronal induction media induced the expression of the neural progenitor marker Sox2 and early stage neuronal markers βIII tubulin and doublecortin.After further culturing in neuronal maintenance media, the induced neuron-like cells also expressed more mature neuronal markers, forkhead box protein P2 (FOXP2), neuronal nuclei protein (NeuN), and the neuronal cell adhesion molecule NCAM1.Although synapsin was also expressed in the induced cells, the level of synapsin expression was lower than that in primary neurons.Moreover, the synaptic vesicle protein SV2 was not detected in the induced neuron-like cells, indicating that they are not fully mature neurons.It is worth noting that the decitabine treatment alone did not induce any neuronal reprogramming.Only the full treatment with decitabine and neuronal induction media followed by neuronal maintenance media allowed the conversion of human skin keratinocytes into neuron-like cells.
More recently, a small molecule cocktail, containing A83-01 (a TGFβ inhibitor), CHIR99021, NaB (a histone deacetylase [HDAC] inhibitor), lysophosphatidic acid (LPA, a phospholipid derivative), rolipram (a PDE4 inhibitor), and SP600125 (a JNK inhibitor) was used in conjunction with the Oct4 transgene, or the Oct4, Sox2, Klf4 and p53 shRNA transgenes, to induce reprogramming of human fibroblasts into neural stem cells (NSCs).The resultant human induced NSCs expressed Pax6, Nestin, and Sox1, and exhibited global gene expression profile similar to that of human embryonic stem cell-derived NSCs.The induced NSCs were also multipotent, displaying the ability to differentiate into neurons, astrocytes and oligodendrocytes.The induced NSC-derived neurons also expressed synaptic protein Synapsin 1 and displayed electrophysiological characteristics of functional neurons.Moreover, they were able to survive and differentiate into neurons and astrocytes in transplanted mouse brains [9].
In a study published soon after, MEFs were induced to reprogram into neural progenitor cells (NPCs) using a small molecule cocktail containing VPA (a HDAC inhibitor), CHIR99021, and RepSox2 (a TGFβ inhibitor), under 5% O 2 , the physiological hypoxic condition [15].The MEF-induced NPCs expressed neural progenitor markers Nestin, Sox2 and Pax6, and could form neurospheres.When cultured under differentiation media, these induced NPCs were able to differentiate into neurons, astrocytes and oligodendrocytes.Neurons derived from the induced NPCs exhibited electrophysiological properties of mature neurons, including repetitive action potential and postsynaptic current.When transplanted into embryonic mouse brains, the induced NPCs were able to give rise to cells that expressed markers of neurons, astrocytes and oligodendrocytes.Mouse neonatal tailtip fibroblasts (TTFs) and human urinary cells could also be reprogrammed into NPC-like cells using the same chemical cocktail and under hypoxic condition.However, whether these NPC-like cells were able to give rise to all three neural lineages in vivo and whether neurons derived from these NPC-like cells can be mature and functional remain to be tested.The mechanisms underlying hypoxia-facilitated reprogramming also remains to be elucidated.
The above studies provide a new paradigm of direct reprogramming by combining transcription factors with small molecule compounds or using small molecules only.Treatment with various small molecule cocktails allowed direct reprogramming of fibroblasts or other somatic cells into neural stem cells or neurons with much higher efficiency, which makes the direct reprogramming strategy much more applicable in regenerative medicine.The replacement of transgenes with small molecules in direct reprogramming would allow a safer and more controllable strategy for cell fate change, further enhancing the therapeutic potential of direct reprogramming.

Small molecules promote direct reprogramming into cardiomyocytes
Direct reprogramming of fibroblasts and other cell types into cardiomyocytes has been investigated relatively heavily in recent years.This sub-field of direct reprogramming has been studied with the intent to apply the findings to treat the loss of cardiomyocytes and impaired heart function, commonly seen after or during heart attacks and heart diseases.Small molecules play a large role in facilitating direct reprogramming into cardiomyocytes, therefore presenting an attempting solution to the inefficiency that often plagues the reprogramming process into cardiomyocytes.
The combination of the small molecule JAK inhibitor JI1 and the cardio-inductive growth factor BMP4, in conjunction with the three pluripotency factors, Oct4, Sox2 and Klf4, induced robust reprogramming from MEFs into cardiomyocytes [16].These induced cardiomyocytes expressed cardiac markers and exhibited spontaneous contraction.Sequential treatment with JI1 and BMP4 increased the efficiency of reprogramming into contracting cardiomyocytes.The efficiency could reach up to 90%.It was proposed that the increase in cardiac induction rate is most likely due to the addition of BMP4, as adding this growth factor alone resulted in a 150-fold increase in contracting patches.JI1 treatment was speculated to promote the expansion of the cardiac precursor pool and inhibit pluripotency [16].
The JAK inhibitor JI1 could also enhance microRNAs-mediated reprogramming of cardiac fibroblasts into functional cardiomyocytes [4].The combination of microRNAs miR-1, miR-133, miR-208, and miR-499 were able to induce direct reprogramming from cardiac fibroblasts into cadiomyocytes at low efficiency.Treatment with the JI1 together with the microRNAs enhanced the efficiency of reprogramming up to 10-fold, presumably through stimulating α-myosin heavy chain (α-MHC) induction and promoting the expression of the L-type calcium channel.The cardiomyocytes induced by JI1 and microRNAs were able to exhibit spontaneous calcium oscillations, indicating they are functional cardiomyocytes [4].
Besides the JAK inhibitor, the TGFβ inhibitor SB431542 was also identified as a small molecule compound to enhance the efficiency of direct reprogramming from MEFs and adult cardiac fibroblasts into cardiomyocytes [6].Selected from a pool of small molecules that have been shown to either promote cardiomyocyte differentiation or iPSC reprogramming, SB431542 was shown to increase the rate of conversion from mouse fibroblasts into cardiomyocytes up to 5-fold, when used together with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5.The small molecules plus transgene-induced cardiomyocytes expressed multiple cardiomyocyte-specific markers, including α-actinin, α-MHC, and myosin light chains, exhibited robust beating.It was proposed that inhibition of TGFβ is likely the reason for the increase in efficiency associated with SB431542 treatment.There was no difference observed in cell proliferation of induced cardiomyocytes between the SB431542 and DMSO control-treated groups, suggesting that SB431542-mediated inhibition of TGFβ signaling plays an active role in the early steps of reprogramming, rather than in the proliferation of induced cells at the later stage [6].
In a more recent study, a small molecule cocktail was identified to induce direct reprogramming of mouse fibroblasts into cardiomyocytes together with the Oct4 transgene [8].The cocktail includes the TGFβ inhibitor SB431542, the GSK3β inhibitor CHIR99021, the LSD1 inhibitor parnate (also called tranylcypromine), and the cAMP activator forskolin.Both MEFs and mouse TTFs were induced into spontaneously contracting cardiomyocytes after being transduced with the Oct4 transgene and treated with the small molecule cocktail.The small molecule treatment is critical for cardiomyocyte reprogramming, because no beating clusters were induced without the compound treatment.The cardiomyocytes induced by the Oct4 transgene and the small molecule combination expressed cardiac-specific markers, such as cardiac troponin T (cTnT) and cardiac myosin heavy chain (cMHC).The TTF-derived cardiomyocytes also exhibited electrophyisiological properties of functional cardiomyocytes, and displayed morphology and characteristics of ventricular-like heart cells.Throughout the conversion process, cells were never observed in a pluripotent state.Instead, the induced cardiomyocytes were derived by passing through a cardiac precursor stage.Although the mechanisms underlying the synergy between the small molecules and the Oct4 transgene remain to be determined, the use of this small molecule cocktail together with the OCT4 transgene to convert fibroblasts into ventricular-like cardiomyocytes nevertheless holds potential for cardiac regenerative medicine, because ventricular cardiomyocytes are commonly lost in heart failure.

Small molecules stimulate direct reprogramming into pancreatic cells
Direct reprogramming into pancreatic cells, especially the insulin-producing β cells, holds great potential for the treatment of diabetes.This sub-field of regenerative medicine has not been investigated as heavily as direct reprogramming into neural cells or cardiomyocytes.However, studies have been performed focusing on directly reprogramming fibroblasts and hepatocytic precursor cells into pancreatic lineages through the use of small molecules and transcription factors.
A combination of small molecules 5-aza-2'-deoxycytidine (decitabine), TSA, retinoic acid (RA, a retinoic acid receptor [RAR] agonist), and a mixture of insulin, transferrin and selenite (ITS) allowed direct reprogramming of rat liver epithelial WB-F344 cells into pancreatic insulin-producing cells in a three-step protocol.In the first stage, stem-like liver epithelial WB-F344 cells were treated with decitabine and TSA, which induced cell morphology change and reduced hepatic marker expression.In the second stage, cells were treated with RA and ITS mix.Over 5% of the cells became pancreatic progenitors expressing Pdx1 at this stage.These progenitor cells were further differentiated into insulin producing cells by treatment with nicotinamide in stage three.At the end of stage three, some induced cells expressed the islet-specific markers Pdx1, NeuroD and insulin I, as well as late-stage pancreatic developmental genes Pax4, Pax6 and MafA.Moreover, the induced cells exhibited glucose-dependent insulin secretion, mimicking islet-like β cells.When the induced progenitor cells generated at stage 2 were transplanted into immunodeficient diabetic nude rats, they were also able to further differentiate into mature insulinproducing cells and improve the hyperglycemia phenotype of the diabetic rats by secreting insulin [17].
In a recent study, inducible expression of the four pluripotency factors, Oct4, Sox2, Klf4, and c-Myc, together with the treatment with Activin A and LiCl, induced MEFs into definitive endoderm-like cells.These cells could differentiate into pancreatic lineages but with low efficiency.Only about 5% and 10% cells expressed Nkx6.1 or Pdx1, individually, and about 1% Nkx6.1 and Pdx1 doublepositive cells, after differentiation.Much more efficient pancreatic induction was obtained when the pluripotency factor-induced definitive endoderm -like cells were treated with a small molecule cocktail containing RA, A83-01 (a TGFβ inhibitor), 2-phospho-Lascorbic acid (pVc, a more stable form of vitamin C), and LDE225 (a Hedgehog pathway inhibitor) [2].Approximately 35% resultant cells expressed Pdx1, about 30% cells expressed Nkx6.1, and about 8% cells expressed both Pdx1 and Nkx6.1, which represents a 2.5 fold, 5 fold, and 7 fold increase, respectively, over cells not treated with the small molecules.The Pdx1 and Nkx6.1 double positive pancreatic progenitor-like cells could be further differentiated into mature pancreatic-like cells by culturing in media containing laminin and nicotinamide [18].Treatment with SB203580 (a MAP kinase inhibitor) and pVc together enhanced the rate of insulinpositive and Pdx1-positive cell population about 4-fold.The insulinpositive cells also expressed Nkx6.1 and Pdx1, secreted insulin and C-peptide in response to stimulation by glucose, tolbutamide and IBMX (3-isobutyl-1-methylxanthine), and ameliorated hyperglycemia in mice induced to become hyperglycemic [2].

Small molecules induce direct reprogramming into myoblasts, chondrocytes, and adipocytes
Small molecule-induced direct reprogramming into mesenchymal cells has been investigated in multiple studies and presents a promising solution to the regeneration or replacement of many different tissue types.The studies in this field could trace back to the seventies.These original studies provided the corner stone for current studies in the field of small molecule-mediated reprogramming.
Direct conversion from one somatic cell type into another by using small molecules has been reported more than thirty years ago.In 1977, it was reported that mouse embryonic nonmuscle C3H/10T1/2 cells could be converted into functional striated muscle cells after treatment with 5-azacytidine (AzaC, a DNMT inhibitor) [19].The multinucleated myotubes exhibited myosin ATPase activity and expressed acetylcholine receptors that could bind the neurotoxin α-bungarotoxin [20].It was proposed that the drug effect is linked to its incorporation into DNA because the deoxy analog 5-aza-2'-deoxycytidine exerts the same effect at even one-tenth of the concentration [20].One year later, the same group showed that other mesenchymal derivatives, such as adipocytes and chondrocytes, besides myotubes, could also be generated from C3H/10T1/2 and Swiss 3T3 cells treated with AzaC [7].The efficiency of reprogramming into both muscle and fat cells was dependent on the concentration of AzaC.High efficiency conversion into adipocytes was also achieved by the treatment with ascorbic acid, therefore its not a unique property of AzaC.The cell type conversion was not resulted from cytotoxicity.It was proposed that the conversion into at least three different cell types of mesenchymal origin could be due to the possibility that cells underwent reprogramming to a more pluripotent state that is confined to mesenchymal lineages, which then differentiate into three individual mesenchymal cell types, although this study could not rule out the possibility that the mouse embryonic 10T½ and 3T3 cell lines contain pluripotent cells, which could be induced to differentiate into the three mesenchymal cell types observed [7].
Reversine, an inhibitor of the Aurora B kinase, was shown to induce mouse myogenic C2C12 cells to acquire multipotent progenitor cell property, having the ability to differentiate into osteogenic and adipogenic cells under relevant stimulation [21].Several other Aurora B inhibitors could also reprogram C2C12 myoblasts in a similar manner [22].About 35% reversinetreated mouse fibroblasts formed multinucleated myotubes, and expressed myogenic markers MHC and MyoD.Reversine-treated mouse fibroblasts could also undergo myogenic differentiation in a transplanted cardiotoxin mouse model, exhibiting the capacity of muscle fiber regeneration.Reversine treatment followed by coculture with C2C12 cells also reprogrammed human fibroblasts into myotubes with an efficiency of 25% [5].It was speculated that Reversine may reprogram somatic cells to a state of increased plasticity so that further stimuli, such as cell-cell interactions, can induce further differentiation with high efficiency.
Recently, the anti-cancer drug 5-nitro-5' hydroxy-indirubin-3' oxime (5'-HNIO) also allowed direct reprogramming skeletal muscle cells into functional adipocytes and osteogenic cells [23].5'-HNIO is an inhibitor of Aurora kinase A, a known regulator of cell fate change.Treatment with 5'-HNIO could reprogram myogenic C2C12 cells into functional adipocytes or osteoblasts at efficiencies similar to that induced by reversine [23].In addition to inducing direct reprogramming within the mesodermal lineage, treatment with 5'-HNIO also allowed direct reprogramming across embryonic lineage boundaries.For example, Treatment with 5'-HNIO could also induced NIH 3T3 cells of ectodermal origin into meseodermal osteogenic cells when the treated cells were cultured with osteogenic factors.Moreover, direct reprogramming induced by 5'-HNIO did not involve the induction of pluripotent gene expression.The lack of pluripotent factor expression suggests that 5'-HNIO treatment could directly reprogram one cell type into another without going through a pluripotent stage, or that 5'-HNIO-treated cells could go through a stage of conditionally reprogrammed cells (CRCs) that do not express pluripotent factors [24].

Conclusions
This review aims to elaborate on the significant roles small molecules play in direct reprogramming and their potential mechanisms of action.As can be seen by the studies outlined above, small molecules have the potential to greatly increase the efficiency and effectiveness of direct reprogramming and even replace transcription factors to induce reprogramming at certain cases.Small molecule-facilitated direct reprogramming is a novel technique that is being exploited as a possible method by which differentiated cells can be produced on a large scale and with high efficiency, while bypassing the pluripotent phase.Not only does this process have the benefit of eliminating the potential problems of tumorigenicity and immunogenicity associated with the use of transgenes, especially the use of oncogenes in iPSC reprogramming, but also is more rapid than inducing iPSCs and then differentiating them into mature, differentiated cells.As scientists investigate this field further, direct reprogramming facilitated by small molecules is bound to become a more common method of not only regenerating lost or damaged tissue, but also as a way to model diseases, screen drugs, and advance the field of medicine as a whole.