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Small molecules and small molecule drugs in regenerative medicine Baisong Lu and Anthony Atala Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA
Regenerative medicine is an emerging, multidisciplinary science that aims to replace or regenerate human cells, tissues or organs, to restore or establish normal function. Research on small molecules and small molecule drugs in regenerative medicine is currently increasing. In this review, we discuss the potential applications of small molecules and small molecule drugs in regenerative medicine. These include enabling novel cell therapy approaches and augmentation of endogenous cells for tissue regeneration, facilitating the generation of target cells for cell therapy, improving the interactions between cells and biomatrices for tissue engineering, and enhancing endogenous stem cell function for tissue regeneration. We also discuss the potential challenges for small molecule drugs in regenerative medicine. Regenerative medicine replaces or regenerates human cells, tissues or organs, to restore or establish normal function [1]. The major distinction between regenerative medicine and traditional molecular medicine is that regenerative medicine restores normal function through the replacement or regeneration of human cells. It involves interdisciplinary research including, but not limited to, tissue engineering, cell therapy, gene therapy and protein pharmaceutics. Small molecule drugs have long been a focus of traditional molecular medicine. Owing to their pharmacological control and ease of use, there have been many recent efforts to search for small molecule drugs for regenerative medicine. In this review, we introduce the application of small molecules and small molecule drugs in regenerative medicine.
Human cells in regenerative medicine
An introduction to regenerative medicine
hiPSCs
Regenerative medicine studies three important components to restore normal function: cells, biomatrices and signaling cues. Whether the goal is to replace nonfunctional or missing cell types, or to regenerate failed organs, human cells are the core component of regenerative medicine. Currently, human pluripotent stem cells {hPSCs [embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)]}, adult stem cells, and terminally differentiated cells are all being studied in regenerative medicine.
Corresponding author: Atala, A. ([email protected]), ([email protected])
hESCs The establishment of hPSCs in 1998 [2] provided a potential limitless cell source for regenerative medicine. With different differentiation protocols, these cells have been differentiated into many cell types. Nonetheless, the use of hESC-differentiated cells in clinical trials has been lagging. Currently, hESC-derived retinal pigmented epithelial cells are being tested to treat Stargardt’s macular dystrophy and dry age-related macular degeneration (AMD) in clinical trials, with the first report published in 2012 [3]. The challenges responsible for this lag are: (i) immune rejection by the host; (ii) the inefficient differentiation and risks of tumorigenicity; and (iii) the lack of efficient cell transplantation methods. To overcome the challenges of immune rejection associated with hESC, hiPSCs were generated by forced expression of transcription factors [4], following Yamanaka’s pioneering work of generating mouse iPSC cells with the transcription factors octamer-binding transcription factor 4/SRY (sex determining region Y)-box 2/Kruppel-like factor 4/C-MYC (OCT4/SOX2/KLF4/C-MYC) [5]. Although there are some differences, hiPSC resemble hESC in that both are pluripotent and have an unlimited capacity for proliferation. Similar to hESC, hiPSC can be induced to differentiate into many cell types, such as neural precursors [6], hepatocytes [7],
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hematopoietic progenitors [8], b cells [9], retinal pigmented epithelial cells [10], skeletal myoblasts [11], motoneurons [12] and cardiomyocytes [13]. Owing to the safety concern of introducing oncogenes (e.g. c-myc) during reprogramming by integrating viral vectors, generating hiPSC using nonintegrating methods (e.g. by using proteins, mRNAs, nonintegrating episomal plasmids and viruses) are available with moderate efficiency. Another strategy is to generate hiPSC with small molecules, which is discussed further below. Recently, the first clinical trial using integration-free hiPSCderived cells to treat AMD was approved in Japan.
Adult stem cells Adult stem cells, also called somatic stem cells, can self-renew and generate the cell types comprising the organs where the stem cells reside. Adult stem cells have been described in the hematopoietic system (hematopoietic stem cells), central nervous system (neural stem cells), liver (liver stem cells), skin (follicle and epidermal stem cells), cornea (the corneal stromal stem cells), gut (intestinal crypt stem cell), skeletal muscle (satellite cells), and adipose tissue and bone marrow (mesenchymal stem cells). Some stem cell types (e.g. amniotic fluid stem cells) are neither hESC/iPSC nor adult stem cells. These cells might also find applications in regenerative medicine owing to their proliferation capacity and differentiation potential. Compared with hESC and hiPSC, adult stem cells have limited proliferation and differentiation capacity in vitro, but they confer a low risk of tumor formation after being transplanted to patients, as shown by the intravenous infusion of human adipose tissuederived mesenchymal stem cells in humans [14]. There are many ongoing clinical trials using adult stem cells to restore normal cell function in various diseases, including neurodegeneration, spinal cord injury, graft-versus-host disease and diabetes. The National Institutes for Health (NIH) database currently registers over 1700 open clinical trials using hematopoietic stem cells, over 100 open clinical trials using mesenchymal stem cells (bone marrow-, cord blood- and adipose-derived) and five open clinical trials using neural stem cells. The US Food and Drug Administration (FDA) has approved several cord blood progenitor cell products for cell therapy; for example, Hemacord from the New York Blood Center, HPC from Clinimmune Labs, and Ducord from Duke University School of Medicine.
Terminally differentiated cells Terminally differentiated cells also have uses in regenerative medicine. For example, fibroblast cells have been used for skin regeneration to treat burns and other ulcerations, and the FDA has approved several products for skin regeneration that contain live fibroblasts [15]. Chondrocytes are being studied to treat osteoarthritis and cartilage defects [16]. Currently, there are more than ten open clinical trials using chondrocytes.
Biomatrices in regenerative medicine Cells actively interact with the biomatrices they contact. In neoorgan engineering, synthetic or natural biomatrices are necessary in addition to cells. Biomatrices serve several important functions: (i) they provide the structural integrity and organizational backbone for cells to organize and assemble; (ii) they mimic the functions of the native extracellular matrix (ECM) to provide support and anchorage for the cells; and (iii) they can be engineered to incorporate bioactive factors to enhance the survival and 2
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guide the differentiation of the cells. For example, neo-bladders engineered and transplanted in patients were generated with polyglycolic acid (PGA) and collagen [17], where PGA provided the rigidity to form the shape of the bladder, and collagen (used to create an ECM) enhanced cellular attachment and growth. There are three types of biomatrix: (i) naturally derived materials, such as collagen, hyaluronic acid (HA), alginate, chitin/chitosan, keratin and silk; (ii) synthetic polymers, such as PGA, polylactic acid (PLA) and poly(-lactic-co-glycolic) acid (PLGA); and (iii) decellularized organ scaffolds. Recently, interest in decellularized organ scaffolds has increased, because these structures preserve the host organ architecture, especially the vascular structure. Acellular matrices from complex organs, such as heart [18], liver [19], lung [20] and kidney [21], have been prepared, and can support cell proliferation and differentiation upon recellularization. Bioengineered airway tissues using decellularized matrix have been transplanted to patients [22,23]. Following this brief introduction to the cells and the biomatrices, the most important components for regenerative medicine, we now discuss the potential applications of small molecules and small molecule drugs in regenerative medicine. We believe that small molecules can be used: (i) in vitro to facilitate the production of target cells and to improve cell survival in cell therapy and tissue engineering; and (ii) in vivo to enhance the proliferation and differentiation of endogenous stem cells to promote tissue regeneration.
Enabling novel cell therapy approaches using small molecules Currently, cell therapy with or without biomaterials is still the major strategy in regenerative medicine. Although the use of small molecule drugs in regenerative medicine is still in an early stage, interest in their use is increasing owing to a better understanding of signaling pathways that control cell fate (especially stem cell fate) and the development of technologies for high-throughput screening (HTS). For a comprehensive review on small molecules regulating the cell fate of various stem cells, readers are referred to a recent review by Schultz et al. [24]. Here, we discuss the applications of small molecules in cell therapy and tissue engineering.
Facilitating the manipulation and maintenance of hESC and/or hiPSC with small molecules Expanding pluripotent hESC and/or hiPSC is crucial for harnessing their therapeutic potential. Spontaneous differentiation is a challenge in hESC and/or hiPSC expansion. Rho-associated protein kinase (ROCK) inhibitor Y-27632 was found to diminish dissociation-induced apoptosis, increase cloning efficiency and facilitate subcloning [25]. The glycogen synthase kinase (GSK)-3b inhibitor 6-bromoindirubin-30 -oxime (BIO) helped to maintain hESC in an undifferentiated state [26]. Many small molecules facilitate the maintenance and proliferation of hESC and/or hiPSC (Table 1) and their use has made these cells more accessible for clinical application. For example, rapamycin and Y-27632 facilitated the largescale suspension expansion of human ESC [27].
Guiding the differentiation of PSCs with small molecules hESC and hiPSC are limitless sources for cell therapy, but pluripotency carries the potential to form teratomas. Although PSCs
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TABLE 1
Small molecules regulating the maintenance and proliferation of PSCs Target cell
Pluripotent stem cell self-renewal and maintenance Bisindolylmaleimide 1i mESC CHIR99021 mESC BIO hESC and mESC hESC and iPSC Y-27632 Thiazovivin Pinacidil SU5402 PD184352 Retinol (vitamin A) Pluripotin/SC1 SB203580 Erythro-9-(2-hydroxy-3-nonyl)adenine
hESC hESC mESC mESC mESC mESC mESC hESC
Pyrintegrin IQ-1
hESC mESC
Small molecules regulating PSC differentiation hESC Purmorphamine CKI-7 hESC and iPSC Y-27632 hESC and iPSC SB-431542 LY294002 CCG143 JAK1 inhibitor 1 Dorsomorphin (Compound C) Verapamil TWS119 Phenazopyridine Cardiogenols (A–D) Isoxazoles IDE1 and 2 Stauprimide
hESC and iPSC hESC and mESC mESC mESC hESC and hiPSC mESC mESC hESC mESC P19 embryonal carcinoma cells mESC mESC and hESC
() Indolactam V
hESC and mESC
Mode of action a Inhibits GSK-3 and enhances ESC self-renewal Inhibits GSK-3 and promotes self-renewal Inhibits GSK-3, activates Wnt signaling and maintains ESC self-renewal Inhibits ROCK, diminishes dissociation-induced apoptosis and increases cloning efficiency Inhibits ROCK and enhances ESC survival Inhibits kinases (e.g. ROCK2, PRK2 and others) and enhances ESC survival Inhibits FGFR receptor and maintains self-renewal Inhibits ERK kinase and maintains self-renewal Activates PI3K and maintains feeder-independent self-renewal Inhibits RasGAP and ERK1, and promotes self-renewal Inhibits P38-MAPK and promotes mESC survival Inhibits adenosine deaminase and phosphodiesterase, and enables hESC maintenance in chemically defined media Activates integrin signaling and improves survival of hESCs upon dissociation Binds to PP2A, decreases phosphorylation of p300 and maintains mouse ES cells in an undifferentiated state Inhibits Shh signaling and promotes neural differentiation Inhibits Wnt, blocks CKI and induces retinal specification Inhibits Rho-associated kinase and induces retinal specification in combination with CKI-7 and SB-431542 Inhibits Nodal and induces neural specification Inhibits PI3K and promotes differentiation to mesoderm Inhibits RhoA signaling and enhances mesoderm differentiation Inhibits JAK1 and enhances mesoderm differentiation Inhibits BMP and promotes neural differentiation Blocks L-type Ca2+ channels and promotes cardiomyocyte differentiation Inhibits GSK-3b and directs neuronal differentiation Mode of action unspecified; enhances neuronal differentiation Mode of action unspecified; efficient induction of cardiogenesis Mode of action unspecified; promotes myocardial differentiation Activates TGF-b signaling and induces endoderm differentiation Inhibits NME2 nuclear localization, downregulates c-Myc, and enhances endoderm, ectoderm and mesoderm differentiation Activates PKC signaling and promotes pancreatic differentiation
a
Abbreviations: BMP, bone morphogenetic protein; CKI, casein kinas inhibitor; ERK, extracellular-signal-regulated kinase; FGFR, fibroblast growth factor receptor; JAK1, Janus kinase 1; MAPK, mitogen-activated protein kinase; NME2, NME/NM23 nucleoside diphosphate kinase 2; P13K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PP2A, protein phosphatase 2; PRK2, protein kinase C-related protein kinase 2; RasGAP, Ras GTPase-activating protein; RhoA, Ras homolog gene family, member A.
can differentiate and integrate into the host organs after transplantation [28], direct transplantation of these cells to patients can carry a risk of tumor formation. Guided differentiation before transplantation can enrich the target cells, which will facilitate downstream application. Thus, PSCs might need to be differentiated into target cells before they can be transplanted safely. Efficient protocols to generate differentiated target cells are necessary to harness the therapeutic potential of hESC and hiPSC for regenerative medicine. Currently, most protocols generating target cells from hESC and/or hiPSC follow the embryonic developmental stages of the target cells through the stepwise use of signaling molecules, most of which are proteins. For example, procedures for guided differentiation toward b cells [29], motoneurons [30] and cardiomyocytes [31] are lengthy, depend on multiple protein growth factors and have relative low yield (Fig. 1). Many small molecules have been found to facilitate the differentiation of PSCs toward various lineages (Table 1). Using small molecules to expedite the process, improve efficiency and (ideally) replace protein factors would
facilitate the eventual clinical use of hESC- and/or hiPSC-derived cells in regenerative medicine. Small molecules have been used in various protocols to facilitate guided differentiation [32,33]. The ultimate goal will be developing guided differentiation protocols using only small molecules.
Facilitating the generation of hiPSC with small molecules Originally, hiPSC generation depended on the expression of various combinations of transcription factors, for example, OCT4/ SOX2/KLF4/MYC [4], OCT4/SOX2/NANOG/LIN28 [34], or OCT4/ SOX2 with or without additional factors. Although integrationfree methods are available, their efficiency is not satisfactory. Small molecules can be used to increase the efficiency and expedite the process of hiPSC generation. For example, the small molecules BIX01294 [35], BayK8644 [36], RepSox [37], valproic acid [38] and vitamin C [39] can increase the efficiency of iPS generation, and enable the omission of one or two transcription factors (Table 2). In addition, Aurora A kinase inhibitor [40], sodium butyrate (a histone deacetylase inhibitor), and SB431542 [a tumor growth
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β cell differentiation
FGF10+ CYC Ex4 + IGF1 + HGF Activin FGF10 + CYC + RA DAPT + Ex4
Activin + Wnt
1–2 d
1–2 d
2–4 d
2–4 d
2–3 d
3d
hESC
β cell
Motoneuron differentiation Reviews POST SCREEN
Neural induction medium
RA
10 d
RA + SHH BDNF + IGF1
7d
3–5 d
~10 d Motoneuron
hESC
Cardiac lineage differentiation Feed depletion
BMP4
BMP4 bFGF Activin A
VEGF DKK1
VEGF DKK1 bFGF
hESC
Cardiomyocytes 2d
1d
3d
4d
6d Drug Discovery Today
FIGURE 1
Examples of differentiation protocols to generate b cells [29], motoneurons [30] and cardiomyocytes [31] from human embryonic stem cells (hESC). The small molecules inducing differentiation are circled. Abbreviations: BDNF, brain-derived neurotrophic factor; BMP4, bone morphogenetic protein 4; CYC, KAADcyclopamine; DAPT, g-secretase inhibitor; DDK1, Dickkopf-1; EB, embryonic body; Ex4, exendin-4; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF1, insulin growth factor 1; RA, all-trans retinoic acid; SHH, Sonic hedgehog; VEGF, vascular endothelial growth factor.
factor (TGF)-b signaling inhibitor] [41] also increase iPSC generation efficiency. A recent study successfully generated mouse iPSC from fibroblasts using only small molecule compounds: valproic acid, CHIR99021, 616452, tranylcypromine, forskolin, 3-deazaneplanocin A and TTNPB [42]. It still remains to be seen whether hiPSCs can be generated in a similar way.
Improving safety in application of target cells derived from PSCs To address the tumorigenicity of PSCs, strategies have been developed to eliminate residual PSCs from differentiated target cells. One strategy is to modify the PSCs so that they express a ‘suicide’ gene [43]. For example, PSCs were engineered to express thymidine kinase by a stem cell-specific promoter; thymidine kinase then transformed the nontoxic small molecule prodrug ganciclovir into the toxic deoxyguanosine triphosphate analog to kill the residual PSCs [44]. More recent work found that isonicotinic acid N0 -phenyl-hydrazine, an inhibitor of stearoyl-coA desaturase, selectively eliminated human PSCs from differentiated cells [45]. This small molecule greatly improves safety but eliminates the need for genetic modification of the PSCs. Glucose-depleted and lactate-enriched culture conditions supported the growth of cardiomyocytes but not other cell types [46]. Using this culture condition to differentiate cardiomyocytes from human PSCs, the authors obtained 99% purity in cardiomyocytes. Thus, glucosedepleted and lactate-enriched culture conditions can facilitate the large-scale production of human cardiomyocytes.
Enhancing recellularization of synthetic and acellular matrix Tissue engineering is an important strategy for regenerative medicine, where synthetic or natural biomatrices are combined with 4
cells to make a neo-organ [17]. With acellular matrices from complex organs, recellularization is necessary to prevent thrombogenesis after transplantation [18–20]. To date, cell reseeding has been achieved through direct cell implantation [17], injection [18] and perfusion [19]. The use of small molecules to enhance cell adhesion, survival and differentiation during recellularization is rare; this might be another field where small molecules can be applied in regenerative medicine. Multipotent stem cells would be a suitable cell source for recellularizing acellular matrices of complex organs, especially those with multiple cell types and limited autologous cells. Small molecules can be used in the perfusate to facilitate the homing and differentiation of multipotent stem cell in acellular organs.
Using small molecule drugs to regulate endogenous stem cell pools and their differentiation Most tissues and organs contain adult stem cells that regenerate damaged cells [47]. Some abnormalities, such as limbal stem cell deficiency in corneal epithelium diseases and hematopoietic stem cell deficiency in bone marrow failure, are caused by the depletion of adult stem cells. Thus, an alternative strategy to cell therapy is to restore the adult stem cell pool so that tissue-specific stem cells can regenerate the missing cell types and restore the function of the tissue.
Activating adult stem cells with small molecule drugs Adult stem cells reside in a microenvironment called the niche, which provides an anatomic space to regulate stem cell number, to instruct stem cells to self-renew or differentiate and to control stem cell motility [47]. Thanks to studies of cell lineage tracing in
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TABLE 2
Name of compound
Target cell
Mode of action
CHIR99021
Rat liver progenitor, MEF and human IMR90 MEF MEF MEF MEF, human keratinocytes MEF MEF Human IMR90 fibroblast Human Keratinocytes Human IMR90 fibroblast, rat liver progenitor cells Rat liver progenitor, human IMR90 MEF miPSCs and hiPSCs Partially reprogrammed cells MEF, NPC IMR90 fibroblasts MEF MEF
Inhibits GSK-3b and supports reprogramming
Forskolin Valproic acid E-616452 (RepSox) Tranylcypromine (Parnate) 3-Deazaneplanocin A (DZNep) TTNPB Sodium butyrate PS-48 A-83-01 PD0325901 Kenpaullone Vitamin C 5-aza-cytidine (AZA) BIX-01294 RG108 (+) Bayk 8644 Suberoylanilide hydroxamic acid (SAHA) Trichostatin A (TSA) SB-431542
MEF MEF and human fibroblasts
Agonist of adenylate cyclase and enables reprogramming in the absence of OCT4 Inhibits HDAC and increases reprogramming efficiency Inhibits TGF-b signaling and replaces Sox2 and c-Myc during reprogramming Inhibits LSD1 and promotes reprogramming in the absence of SOX2 Inhibits lysine MTase EZH2 and facilitates late reprogramming A retinoic acid receptor ligand that enhances chemical reprogramming efficiency Inhibits histone acetylase and improves reprogramming efficiency Activates PDK1, enhancing reprogramming efficiency Inhibits TGF-b and promotes reprogramming of human and rat cells Inhibits MEK and supports reprogramming Inhibits GSK-3b and replaces Klf4 Enhances reprogramming efficiency Inhibits DNA MTase and induces fully reprogrammed iPS state Inhibits G9a histone MTase and increases reprogramming efficiency Inhibits DNA MTase and facilitates reprogramming of fibroblasts L-channel calcium agonist, supports reprogramming of MEF Inhibits HDAC and improves MEF reprogramming efficiency
Thiazovivin
Human fibroblasts
AMI-5
MEF
8-Bromoadenosine 30 ,50 -cyclic monophosphate (8-BrcAMP) Dasatinib PP1 iPYrazine (iPY)
Human foreskin fibroblasts
Inhibits HDAC and improves MEF reprogramming efficiency with four iPSC TFs Inhibits TGF-b receptor, enhances MEF reprogramming efficiency in the absence of c-Myc or Sox2, and enhances and accelerates reprogramming of human somatic cells Inhibits ROCK, and enhances and accelerates reprogramming of human somatic cells Inhibits protein arginine MTase, in combination with A-83-01, enables reprogramming of MEFs with Oct4 only Activates PKA and improves reprogramming efficiency
MEF MEF MEF
Src family kinase inhibitor, replaces Sox2 in MEF reprogramming Src family kinase inhibitor, replaces Sox2 in MEF reprogramming Src family kinase inhibitor, replaces Sox2 in MEF reprogramming
a
The seven small molecules combined that can fully reprogram mouse fibroblasts [42] are highlighted in italic type. Abbreviations: EZH2, enhancer of zeste homolog 2; HDAC, histone deacetylase; LSD1, lysine-specific demethylase 1; MEF, mouse embryonic fibroblast; MEK, mitogen-activated protein kinase kinase; MTase, methyltransferase; PDK1, phosphoinositide-dependent kinase-1; PKA, protein kinase A; TF, transcription factor. b
transgenic mice, the signaling pathways [e.g. the Wnt signaling, the Sonic hedgehog (SHH) signaling and the TGF-b signaling pathways] regulating various adult stem cells are beginning to be revealed. There is good reason to believe that human adult stem cells are similar, although species-specific differences might exist. This knowledge makes enhancing tissue regeneration through regulating endogenous adult stem cells an attractive strategy. Many genetic studies have demonstrated that impairing signaling pathways that are important to adult stem cell maintenance results in deletion of the adult stem cell pool [48,49]. In regenerative medicine, it is desirable to enhance the adult stem cell pool through manipulating these important signaling pathways. For example, erythropoietin infused into the brains of mice increased production of neural progenitors from neural stem cells [50]. Mildly increasing Wnt signaling levels through genetic manipulation enhanced hematopoietic stem cell function [51]. Although protein infusion to the brain and genetic manipulation are impractical in humans, regulating adult stem cells with small molecule drugs is likely to become feasible. An encouraging example is the activation of SHH signaling to stimulate hair generation in mouse skin through one topical application of a small molecule SHH agonist [52].
Overactivation of Wnt, SHH and Notch signaling is associated with tumor formation and, currently, there are many studies searching for inhibitors of these signaling pathways to explore their potential in cancer therapy. Fewer studies have reported activators (agonists) of these signaling pathways. Reasons for this bias include the overwhelming need and interest in finding drugs to eradicate cancer, and the concern relating to tumorigenic risks of the signaling pathway activators. It is unclear whether these signaling pathways are intrinsically easy to inhibit but hard to activate. The risks of tumorigenicity from small molecule drugs activating adult stem cells could be controlled by transient and local drug delivery. This can be achieved by controlled release of small molecule drugs by implanted scaffolds and injectable gels [53]. In addition, the chance of cancerous cell formation from transient stem cell activation is unclear at present. Furthermore, those patients being treated might have stem cell deficiency. Thus, in the future, the tumorigenic risk of activating adult stem cells by small molecule drugs for tissue regeneration might not be such a formidable challenge. Some small molecules regulate adult stem cells, mainly hematopoietic stem cells, neural stem cells and mesenchymal stem cells (reviewed in [54]). Tetraethylenepentamine (Gamida Cell Ltd) and 16,16-dimethylpro-staglandin E2
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TABLE 3
Examples of cell fate changea,b Starting cell Genetic approaches b cells Amniotic cells Exocrine cells Fibroblasts Reviews POST SCREEN
Human fibroblasts Myelomonocytes Small molecule-induced changes Annulus fibrosus cells C2C12
Target cell
Transcription factors and/or chemicals
Macrophages Vascular endothelial cells b-cells Myoblasts Hepatocytes Neuron Cardiomyocyte Blood progenitors Neural cells Cardiomyocytes Angioblast-like progenitors Cholinergic neurons Neuron Erythroid-megakaryocytic cells, eosinophils
C/EBPa/C/EBPb ETV2 and other factors PDX1/NGN3/MAFA MyoD HNF1a/FOXA3/GATA4; HNF4a/FOXA1 (or FOXA2 or FOXA3) ASCL1/BRN2/MYT1L; or miRNAs; ASCL1/NURR1/LMX1A GATA4/MEF2C/TBX5; GATA4/BAF60C/TBX5; GATA4/HAND2/MEF2C/TBX5 OCT4 OCT4/SOX2/KLF4/C-MYC OCT4/SOX2/KLF4/C-MYC OCT4/SOX2/KLF4/C-MYC NGN2, facilitated by small molecules forskolin and dorsomorphin ASCL1, NGN2, facilitated by small molecules SB431542 and Chir99021 GATA1
Skeletal progenitor cells Multipotent mesenchymal progenitors
Reversine (MEK1/NMMII/Aurora inhibitor) Reversine (MEK1/NMMII/Aurora inhibitor) Hesperadin (Aurora inhibitor) VX-680 (Aurora inhibitor) MLN-8054 (Aurora inhibitor) PD173074/PD0325901/Chir99021/A-83-01/Parnate Butyrate (HDAC inhibitor) Trichostatin A (HDAC inhibitor) MS-275 (HDAC inhibitor) Apicidin (HDAC inhibitor) BRD7389 (inhibitor of ribosomal S6 kinases) Myoseverin (binding to microtubules) Dexamethasone (inducing C/EBPbeta)
EpiSC Oligodendrocyte precursor cells
Multipotent progenitors mESC Multipotent neural stem cells
Mouse a-cell line aTC1 Myotubes Pancreatic cells
b-cell Mononucleotide cells Hepatocytes
a
Please refer to Table S1 in the supplementary material online for references relevant to this table. Abbreviations: ASCL1, Achaete-scute homolog 1; C/EBP, CCAAT/enhancer binding protein; ETV2, ets variant 2; EZH2 (NMMII), enhancer of zeste homolog 2; FOXA3, forkhead box A3; GATA1, GATA binding protein 1; HAND2, heart and neural crest derivatives expressed; HDAC, histone deacetylase; HNF, hepatocyte nuclear factor; LMX1a, LIM homeobox transcription factor 1, alpha; MEK, mitogen-activated protein kinase kinase; MYTL1, myelin transcription factor 1-like; NGN, neurogenin; NURR1, nuclear receptor related 1 protein; PDX1, pancreatic and duodenal homeobox 1; TBX5, T-box transcription factor. b
(FT1050, Fate Therapeutics) have been studied for their activity to improve the proliferation and function of cord blood cells ex vivo.
Generating target cells by small molecule induced cell fate conversion Using genetic strategies, it is now possible to change cells directly from one cell fate (mostly fibroblasts) into another, a process called transdifferentiation (Table 3). It is evident that fibroblasts can be converted into cells from all three germ layers. Two strategies have been used: (i) forced expression or ablation of lineage-specific regulators, such as transcription factors and miRNAs (highlighted in Table 3); and (ii) timed, forced expression of iPSC-reprogramming factors, combined with lineage-specific culturing conditions [55]. Although most work on cell fate conversion was performed in vitro, endogenous exocrine cells can be transformed into b cells in vivo [56], and murine cardiac fibroblasts can be reprogrammed into cardiomyocytes by virus-mediated transcription factor expression in vivo [57]. Fibroblasts are abundant in many tissues. The ability to transform fibroblasts directly into the missing cell type important to the normal function of an organ (e.g. myocytes in skeletal muscle, cardiomyocytes in the heart, neurons in the brain and hepatocytes in the liver) will facilitate the restoration of normal function. 6
Currently, cell fate conversions are mostly achieved by genetic approaches that upregulate or inhibit genes that regulate cell fates. To eliminate safety concerns, it is hoped that small molecule drugs might be able to change the existing cells in situ into the cell types that are missing. Small molecule-induced cell fate changes have been described, although in most cases the process is de-differentiation (changing from a more differentiated state to a less differentiated state) (Table 3). With the exception of pancreatic to hepatic conversion in some pathological conditions, most chemical-induced cell fate changes have been observed in vitro. In regenerative medicine, two approaches could be adopted for small molecule drug-mediated trans-differentiation in vivo. First, small molecules controlling the expression of lineage-specific regulators can be searched for in terms of their capacity to change cell fate, which can then be tested. Second, timed treatments with iPSC reprogramming chemicals [42] could be tested for their ability to change cell fate in vivo. Although it is difficult to control the cell environment in vivo to favor cell fate conversion into a specific lineage, this might not be an issue because each organ would be the best environment for the differentiation of its resident cells. This view is supported by the observation that the in vivo reprogramming of fibroblasts into cardiomyocytes by transcription factors was more complete than
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the in vitro process [57]. However, several questions still remain: for example, how much of the in vitro work can be replicated in vivo? How tightly can this process be controlled? When a given disease is present, how can the cell environment be made favorable for cell fate conversion?
Screening for small molecules with potential use in regenerative medicine Although biochemical HTS has been widely used in the pharmaceutical industry, cell-based HTS is frequently used to screen for small molecules that could be useful in regenerative medicine. Most small molecules listed in this review were found by screening using PSCs, mesenchymal stem cells, neural stem cells and hematopoietic stem cells, which are readily available. Cell-based HTS using other types of adult stem cell has been difficult because of the limited availability of these cells. Zebrafish is a suitable system amenable to HTS for small molecules regulating adult stem cells and 16,16-dimethylpro-staglandin E2 (FT1050) was found by using this system [58]. Intestinal organoids [59] and liver organoids [60] culturing protocols have been developed that might make screening for small molecules regulating intestine and liver stem cells possible.
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inhibitor, Italfarmaco) and HT-100/halofuginone (Halo Therapeutics, LLC) for promoting muscle regeneration in patients with Duchenne muscular dystrophy. Currently, hESCs and hiPSCs have garnered the most attention for their potential in regenerative medicine, and small molecules modulating these cells are being actively investigated. Small molecules regulating adult stem cells in vivo are likely to find wide application in regenerative medicine, and HTS with organoids might help to find such small molecules. At present, little attention is being paid to the use of small molecules to enhance cell–matrix interactions. However, with interest in acellular matrices increasing, research into this area is also likely to increase. There are still many challenges in the development of chemical leads into drugs for regenerative medicine. One is that the in vitro performance of a small molecule does not necessarily predict its in vivo performance. For example, isoxazole showed promising cardiogenic activity in vitro, but had no beneficial effects in myocardial infarction models. In addition, the tumorigenic risk of activating endogenous stem cells with small molecules to enhance tissue regeneration needs to be determined. Despite these challenges and questions, small molecules and small molecule drugs are certain to have an increasingly important role in regenerative medicine.
Current status and future directions Although currently small molecule drugs meeting the definition of regenerative medicine are still under development, this situation is likely to change in the future. Several small molecule drugs in clinical trials can be regarded as regenerative medicine. These include nitric oxide-releasing gel (sponsored by China Medical University Hospital) for promoting hair regeneration in subjects with androgenic alopecia, and Givinostat (a histone deacetylase
Acknowledgement The authors thank Karen Klein for editing the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.drudis. 2013.11.011.
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44 Zhao, Q. et al. (2012) Safeguarding pluripotent stem cells for cell therapy with a nonviral, non-integrating episomal suicide construct. Biomaterials 33, 7261–7271 45 Ben-David, U. et al. (2013) Selective elimination of human pluripotent stem cells by an oleate synthesis inhibitor discovered in a high-throughput screen. Cell Stem Cell 12, 167–179 46 Tohyama, S. et al. (2013) Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127–137 47 Morrison, S.J. and Spradling, A.C. (2008) Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 132, 598–611 48 Korinek, V. et al. (1998) Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat. Genet. 19, 379–383 49 Soucek, L. et al. (2008) Modelling Myc inhibition as a cancer therapy. Nature 455, 679–683 50 Shingo, T. et al. (2001) Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J. Neurosci. 21, 9733–9743 51 Luis, T.C. et al. (2011) Canonical wnt signaling regulates hematopoiesis in a dosagedependent fashion. Cell Stem Cell 9, 345–356 52 Paladini, R.D. et al. (2005) Modulation of hair growth with small molecule agonists of the hedgehog signaling pathway. J. Invest. Dermatol 125, 638–646 53 Sternberg, K. et al. (2013) Implant-associated local drug delivery systems based on biodegradable polymers: customized designs for different medical applications. Biomed. Tech. 58, 417–427 54 Lairson, L.L. et al. (2013) Small molecule-based approaches to adult stem cell therapies. Annu. Rev. Pharmacol. Toxicol. 53, 107–125 55 Ma, T. et al. (2013) Progress in the reprogramming of somatic cells. Circ. Res. 112, 562–574 56 Zhou, Q. et al. (2008) In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature 455, 627–632 57 Qian, L. et al. (2012) In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598 58 North, T.E. et al. (2007) Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 447, 1007–1011 59 Kuratnik, A. and Giardina, C. (2013) Intestinal organoids as tissue surrogates for toxicological and pharmacological studies. Biochem. Pharmacol. 85, 1721–1726 60 Huch, M. et al. (2013) In vitro expansion of single Lgr51 liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250
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Small molecules and small molecule drugs in regenerative medicine Baisong Lu and Anthony Atala Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA
Regenerative medicine is an emerging, multidisciplinary science that aims to replace or regenerate human cells, tissues or organs, to restore or establish normal function. Research on small molecules and small molecule drugs in regenerative medicine is currently increasing. In this review, we discuss the potential applications of small molecules and small molecule drugs in regenerative medicine. These include enabling novel cell therapy approaches and augmentation of endogenous cells for tissue regeneration, facilitating the generation of target cells for cell therapy, improving the interactions between cells and biomatrices for tissue engineering, and enhancing endogenous stem cell function for tissue regeneration. We also discuss the potential challenges for small molecule drugs in regenerative medicine. Regenerative medicine replaces or regenerates human cells, tissues or organs, to restore or establish normal function [1]. The major distinction between regenerative medicine and traditional molecular medicine is that regenerative medicine restores normal function through the replacement or regeneration of human cells. It involves interdisciplinary research including, but not limited to, tissue engineering, cell therapy, gene therapy and protein pharmaceutics. Small molecule drugs have long been a focus of traditional molecular medicine. Owing to their pharmacological control and ease of use, there have been many recent efforts to search for small molecule drugs for regenerative medicine. In this review, we introduce the application of small molecules and small molecule drugs in regenerative medicine.
Human cells in regenerative medicine
An introduction to regenerative medicine
hiPSCs
Regenerative medicine studies three important components to restore normal function: cells, biomatrices and signaling cues. Whether the goal is to replace nonfunctional or missing cell types, or to regenerate failed organs, human cells are the core component of regenerative medicine. Currently, human pluripotent stem cells {hPSCs [embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)]}, adult stem cells, and terminally differentiated cells are all being studied in regenerative medicine.
Corresponding author: Atala, A. ([email protected]), ([email protected])
hESCs The establishment of hPSCs in 1998 [2] provided a potential limitless cell source for regenerative medicine. With different differentiation protocols, these cells have been differentiated into many cell types. Nonetheless, the use of hESC-differentiated cells in clinical trials has been lagging. Currently, hESC-derived retinal pigmented epithelial cells are being tested to treat Stargardt’s macular dystrophy and dry age-related macular degeneration (AMD) in clinical trials, with the first report published in 2012 [3]. The challenges responsible for this lag are: (i) immune rejection by the host; (ii) the inefficient differentiation and risks of tumorigenicity; and (iii) the lack of efficient cell transplantation methods. To overcome the challenges of immune rejection associated with hESC, hiPSCs were generated by forced expression of transcription factors [4], following Yamanaka’s pioneering work of generating mouse iPSC cells with the transcription factors octamer-binding transcription factor 4/SRY (sex determining region Y)-box 2/Kruppel-like factor 4/C-MYC (OCT4/SOX2/KLF4/C-MYC) [5]. Although there are some differences, hiPSC resemble hESC in that both are pluripotent and have an unlimited capacity for proliferation. Similar to hESC, hiPSC can be induced to differentiate into many cell types, such as neural precursors [6], hepatocytes [7],
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hematopoietic progenitors [8], b cells [9], retinal pigmented epithelial cells [10], skeletal myoblasts [11], motoneurons [12] and cardiomyocytes [13]. Owing to the safety concern of introducing oncogenes (e.g. c-myc) during reprogramming by integrating viral vectors, generating hiPSC using nonintegrating methods (e.g. by using proteins, mRNAs, nonintegrating episomal plasmids and viruses) are available with moderate efficiency. Another strategy is to generate hiPSC with small molecules, which is discussed further below. Recently, the first clinical trial using integration-free hiPSCderived cells to treat AMD was approved in Japan.
Adult stem cells Adult stem cells, also called somatic stem cells, can self-renew and generate the cell types comprising the organs where the stem cells reside. Adult stem cells have been described in the hematopoietic system (hematopoietic stem cells), central nervous system (neural stem cells), liver (liver stem cells), skin (follicle and epidermal stem cells), cornea (the corneal stromal stem cells), gut (intestinal crypt stem cell), skeletal muscle (satellite cells), and adipose tissue and bone marrow (mesenchymal stem cells). Some stem cell types (e.g. amniotic fluid stem cells) are neither hESC/iPSC nor adult stem cells. These cells might also find applications in regenerative medicine owing to their proliferation capacity and differentiation potential. Compared with hESC and hiPSC, adult stem cells have limited proliferation and differentiation capacity in vitro, but they confer a low risk of tumor formation after being transplanted to patients, as shown by the intravenous infusion of human adipose tissuederived mesenchymal stem cells in humans [14]. There are many ongoing clinical trials using adult stem cells to restore normal cell function in various diseases, including neurodegeneration, spinal cord injury, graft-versus-host disease and diabetes. The National Institutes for Health (NIH) database currently registers over 1700 open clinical trials using hematopoietic stem cells, over 100 open clinical trials using mesenchymal stem cells (bone marrow-, cord blood- and adipose-derived) and five open clinical trials using neural stem cells. The US Food and Drug Administration (FDA) has approved several cord blood progenitor cell products for cell therapy; for example, Hemacord from the New York Blood Center, HPC from Clinimmune Labs, and Ducord from Duke University School of Medicine.
Terminally differentiated cells Terminally differentiated cells also have uses in regenerative medicine. For example, fibroblast cells have been used for skin regeneration to treat burns and other ulcerations, and the FDA has approved several products for skin regeneration that contain live fibroblasts [15]. Chondrocytes are being studied to treat osteoarthritis and cartilage defects [16]. Currently, there are more than ten open clinical trials using chondrocytes.
Biomatrices in regenerative medicine Cells actively interact with the biomatrices they contact. In neoorgan engineering, synthetic or natural biomatrices are necessary in addition to cells. Biomatrices serve several important functions: (i) they provide the structural integrity and organizational backbone for cells to organize and assemble; (ii) they mimic the functions of the native extracellular matrix (ECM) to provide support and anchorage for the cells; and (iii) they can be engineered to incorporate bioactive factors to enhance the survival and 2
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guide the differentiation of the cells. For example, neo-bladders engineered and transplanted in patients were generated with polyglycolic acid (PGA) and collagen [17], where PGA provided the rigidity to form the shape of the bladder, and collagen (used to create an ECM) enhanced cellular attachment and growth. There are three types of biomatrix: (i) naturally derived materials, such as collagen, hyaluronic acid (HA), alginate, chitin/chitosan, keratin and silk; (ii) synthetic polymers, such as PGA, polylactic acid (PLA) and poly(-lactic-co-glycolic) acid (PLGA); and (iii) decellularized organ scaffolds. Recently, interest in decellularized organ scaffolds has increased, because these structures preserve the host organ architecture, especially the vascular structure. Acellular matrices from complex organs, such as heart [18], liver [19], lung [20] and kidney [21], have been prepared, and can support cell proliferation and differentiation upon recellularization. Bioengineered airway tissues using decellularized matrix have been transplanted to patients [22,23]. Following this brief introduction to the cells and the biomatrices, the most important components for regenerative medicine, we now discuss the potential applications of small molecules and small molecule drugs in regenerative medicine. We believe that small molecules can be used: (i) in vitro to facilitate the production of target cells and to improve cell survival in cell therapy and tissue engineering; and (ii) in vivo to enhance the proliferation and differentiation of endogenous stem cells to promote tissue regeneration.
Enabling novel cell therapy approaches using small molecules Currently, cell therapy with or without biomaterials is still the major strategy in regenerative medicine. Although the use of small molecule drugs in regenerative medicine is still in an early stage, interest in their use is increasing owing to a better understanding of signaling pathways that control cell fate (especially stem cell fate) and the development of technologies for high-throughput screening (HTS). For a comprehensive review on small molecules regulating the cell fate of various stem cells, readers are referred to a recent review by Schultz et al. [24]. Here, we discuss the applications of small molecules in cell therapy and tissue engineering.
Facilitating the manipulation and maintenance of hESC and/or hiPSC with small molecules Expanding pluripotent hESC and/or hiPSC is crucial for harnessing their therapeutic potential. Spontaneous differentiation is a challenge in hESC and/or hiPSC expansion. Rho-associated protein kinase (ROCK) inhibitor Y-27632 was found to diminish dissociation-induced apoptosis, increase cloning efficiency and facilitate subcloning [25]. The glycogen synthase kinase (GSK)-3b inhibitor 6-bromoindirubin-30 -oxime (BIO) helped to maintain hESC in an undifferentiated state [26]. Many small molecules facilitate the maintenance and proliferation of hESC and/or hiPSC (Table 1) and their use has made these cells more accessible for clinical application. For example, rapamycin and Y-27632 facilitated the largescale suspension expansion of human ESC [27].
Guiding the differentiation of PSCs with small molecules hESC and hiPSC are limitless sources for cell therapy, but pluripotency carries the potential to form teratomas. Although PSCs
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TABLE 1
Small molecules regulating the maintenance and proliferation of PSCs Target cell
Pluripotent stem cell self-renewal and maintenance Bisindolylmaleimide 1i mESC CHIR99021 mESC BIO hESC and mESC hESC and iPSC Y-27632 Thiazovivin Pinacidil SU5402 PD184352 Retinol (vitamin A) Pluripotin/SC1 SB203580 Erythro-9-(2-hydroxy-3-nonyl)adenine
hESC hESC mESC mESC mESC mESC mESC hESC
Pyrintegrin IQ-1
hESC mESC
Small molecules regulating PSC differentiation hESC Purmorphamine CKI-7 hESC and iPSC Y-27632 hESC and iPSC SB-431542 LY294002 CCG143 JAK1 inhibitor 1 Dorsomorphin (Compound C) Verapamil TWS119 Phenazopyridine Cardiogenols (A–D) Isoxazoles IDE1 and 2 Stauprimide
hESC and iPSC hESC and mESC mESC mESC hESC and hiPSC mESC mESC hESC mESC P19 embryonal carcinoma cells mESC mESC and hESC
() Indolactam V
hESC and mESC
Mode of action a Inhibits GSK-3 and enhances ESC self-renewal Inhibits GSK-3 and promotes self-renewal Inhibits GSK-3, activates Wnt signaling and maintains ESC self-renewal Inhibits ROCK, diminishes dissociation-induced apoptosis and increases cloning efficiency Inhibits ROCK and enhances ESC survival Inhibits kinases (e.g. ROCK2, PRK2 and others) and enhances ESC survival Inhibits FGFR receptor and maintains self-renewal Inhibits ERK kinase and maintains self-renewal Activates PI3K and maintains feeder-independent self-renewal Inhibits RasGAP and ERK1, and promotes self-renewal Inhibits P38-MAPK and promotes mESC survival Inhibits adenosine deaminase and phosphodiesterase, and enables hESC maintenance in chemically defined media Activates integrin signaling and improves survival of hESCs upon dissociation Binds to PP2A, decreases phosphorylation of p300 and maintains mouse ES cells in an undifferentiated state Inhibits Shh signaling and promotes neural differentiation Inhibits Wnt, blocks CKI and induces retinal specification Inhibits Rho-associated kinase and induces retinal specification in combination with CKI-7 and SB-431542 Inhibits Nodal and induces neural specification Inhibits PI3K and promotes differentiation to mesoderm Inhibits RhoA signaling and enhances mesoderm differentiation Inhibits JAK1 and enhances mesoderm differentiation Inhibits BMP and promotes neural differentiation Blocks L-type Ca2+ channels and promotes cardiomyocyte differentiation Inhibits GSK-3b and directs neuronal differentiation Mode of action unspecified; enhances neuronal differentiation Mode of action unspecified; efficient induction of cardiogenesis Mode of action unspecified; promotes myocardial differentiation Activates TGF-b signaling and induces endoderm differentiation Inhibits NME2 nuclear localization, downregulates c-Myc, and enhances endoderm, ectoderm and mesoderm differentiation Activates PKC signaling and promotes pancreatic differentiation
a
Abbreviations: BMP, bone morphogenetic protein; CKI, casein kinas inhibitor; ERK, extracellular-signal-regulated kinase; FGFR, fibroblast growth factor receptor; JAK1, Janus kinase 1; MAPK, mitogen-activated protein kinase; NME2, NME/NM23 nucleoside diphosphate kinase 2; P13K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PP2A, protein phosphatase 2; PRK2, protein kinase C-related protein kinase 2; RasGAP, Ras GTPase-activating protein; RhoA, Ras homolog gene family, member A.
can differentiate and integrate into the host organs after transplantation [28], direct transplantation of these cells to patients can carry a risk of tumor formation. Guided differentiation before transplantation can enrich the target cells, which will facilitate downstream application. Thus, PSCs might need to be differentiated into target cells before they can be transplanted safely. Efficient protocols to generate differentiated target cells are necessary to harness the therapeutic potential of hESC and hiPSC for regenerative medicine. Currently, most protocols generating target cells from hESC and/or hiPSC follow the embryonic developmental stages of the target cells through the stepwise use of signaling molecules, most of which are proteins. For example, procedures for guided differentiation toward b cells [29], motoneurons [30] and cardiomyocytes [31] are lengthy, depend on multiple protein growth factors and have relative low yield (Fig. 1). Many small molecules have been found to facilitate the differentiation of PSCs toward various lineages (Table 1). Using small molecules to expedite the process, improve efficiency and (ideally) replace protein factors would
facilitate the eventual clinical use of hESC- and/or hiPSC-derived cells in regenerative medicine. Small molecules have been used in various protocols to facilitate guided differentiation [32,33]. The ultimate goal will be developing guided differentiation protocols using only small molecules.
Facilitating the generation of hiPSC with small molecules Originally, hiPSC generation depended on the expression of various combinations of transcription factors, for example, OCT4/ SOX2/KLF4/MYC [4], OCT4/SOX2/NANOG/LIN28 [34], or OCT4/ SOX2 with or without additional factors. Although integrationfree methods are available, their efficiency is not satisfactory. Small molecules can be used to increase the efficiency and expedite the process of hiPSC generation. For example, the small molecules BIX01294 [35], BayK8644 [36], RepSox [37], valproic acid [38] and vitamin C [39] can increase the efficiency of iPS generation, and enable the omission of one or two transcription factors (Table 2). In addition, Aurora A kinase inhibitor [40], sodium butyrate (a histone deacetylase inhibitor), and SB431542 [a tumor growth
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β cell differentiation
FGF10+ CYC Ex4 + IGF1 + HGF Activin FGF10 + CYC + RA DAPT + Ex4
Activin + Wnt
1–2 d
1–2 d
2–4 d
2–4 d
2–3 d
3d
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Neural induction medium
RA
10 d
RA + SHH BDNF + IGF1
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3–5 d
~10 d Motoneuron
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Cardiac lineage differentiation Feed depletion
BMP4
BMP4 bFGF Activin A
VEGF DKK1
VEGF DKK1 bFGF
hESC
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FIGURE 1
Examples of differentiation protocols to generate b cells [29], motoneurons [30] and cardiomyocytes [31] from human embryonic stem cells (hESC). The small molecules inducing differentiation are circled. Abbreviations: BDNF, brain-derived neurotrophic factor; BMP4, bone morphogenetic protein 4; CYC, KAADcyclopamine; DAPT, g-secretase inhibitor; DDK1, Dickkopf-1; EB, embryonic body; Ex4, exendin-4; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF1, insulin growth factor 1; RA, all-trans retinoic acid; SHH, Sonic hedgehog; VEGF, vascular endothelial growth factor.
factor (TGF)-b signaling inhibitor] [41] also increase iPSC generation efficiency. A recent study successfully generated mouse iPSC from fibroblasts using only small molecule compounds: valproic acid, CHIR99021, 616452, tranylcypromine, forskolin, 3-deazaneplanocin A and TTNPB [42]. It still remains to be seen whether hiPSCs can be generated in a similar way.
Improving safety in application of target cells derived from PSCs To address the tumorigenicity of PSCs, strategies have been developed to eliminate residual PSCs from differentiated target cells. One strategy is to modify the PSCs so that they express a ‘suicide’ gene [43]. For example, PSCs were engineered to express thymidine kinase by a stem cell-specific promoter; thymidine kinase then transformed the nontoxic small molecule prodrug ganciclovir into the toxic deoxyguanosine triphosphate analog to kill the residual PSCs [44]. More recent work found that isonicotinic acid N0 -phenyl-hydrazine, an inhibitor of stearoyl-coA desaturase, selectively eliminated human PSCs from differentiated cells [45]. This small molecule greatly improves safety but eliminates the need for genetic modification of the PSCs. Glucose-depleted and lactate-enriched culture conditions supported the growth of cardiomyocytes but not other cell types [46]. Using this culture condition to differentiate cardiomyocytes from human PSCs, the authors obtained 99% purity in cardiomyocytes. Thus, glucosedepleted and lactate-enriched culture conditions can facilitate the large-scale production of human cardiomyocytes.
Enhancing recellularization of synthetic and acellular matrix Tissue engineering is an important strategy for regenerative medicine, where synthetic or natural biomatrices are combined with 4
cells to make a neo-organ [17]. With acellular matrices from complex organs, recellularization is necessary to prevent thrombogenesis after transplantation [18–20]. To date, cell reseeding has been achieved through direct cell implantation [17], injection [18] and perfusion [19]. The use of small molecules to enhance cell adhesion, survival and differentiation during recellularization is rare; this might be another field where small molecules can be applied in regenerative medicine. Multipotent stem cells would be a suitable cell source for recellularizing acellular matrices of complex organs, especially those with multiple cell types and limited autologous cells. Small molecules can be used in the perfusate to facilitate the homing and differentiation of multipotent stem cell in acellular organs.
Using small molecule drugs to regulate endogenous stem cell pools and their differentiation Most tissues and organs contain adult stem cells that regenerate damaged cells [47]. Some abnormalities, such as limbal stem cell deficiency in corneal epithelium diseases and hematopoietic stem cell deficiency in bone marrow failure, are caused by the depletion of adult stem cells. Thus, an alternative strategy to cell therapy is to restore the adult stem cell pool so that tissue-specific stem cells can regenerate the missing cell types and restore the function of the tissue.
Activating adult stem cells with small molecule drugs Adult stem cells reside in a microenvironment called the niche, which provides an anatomic space to regulate stem cell number, to instruct stem cells to self-renew or differentiate and to control stem cell motility [47]. Thanks to studies of cell lineage tracing in
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TABLE 2
Name of compound
Target cell
Mode of action
CHIR99021
Rat liver progenitor, MEF and human IMR90 MEF MEF MEF MEF, human keratinocytes MEF MEF Human IMR90 fibroblast Human Keratinocytes Human IMR90 fibroblast, rat liver progenitor cells Rat liver progenitor, human IMR90 MEF miPSCs and hiPSCs Partially reprogrammed cells MEF, NPC IMR90 fibroblasts MEF MEF
Inhibits GSK-3b and supports reprogramming
Forskolin Valproic acid E-616452 (RepSox) Tranylcypromine (Parnate) 3-Deazaneplanocin A (DZNep) TTNPB Sodium butyrate PS-48 A-83-01 PD0325901 Kenpaullone Vitamin C 5-aza-cytidine (AZA) BIX-01294 RG108 (+) Bayk 8644 Suberoylanilide hydroxamic acid (SAHA) Trichostatin A (TSA) SB-431542
MEF MEF and human fibroblasts
Agonist of adenylate cyclase and enables reprogramming in the absence of OCT4 Inhibits HDAC and increases reprogramming efficiency Inhibits TGF-b signaling and replaces Sox2 and c-Myc during reprogramming Inhibits LSD1 and promotes reprogramming in the absence of SOX2 Inhibits lysine MTase EZH2 and facilitates late reprogramming A retinoic acid receptor ligand that enhances chemical reprogramming efficiency Inhibits histone acetylase and improves reprogramming efficiency Activates PDK1, enhancing reprogramming efficiency Inhibits TGF-b and promotes reprogramming of human and rat cells Inhibits MEK and supports reprogramming Inhibits GSK-3b and replaces Klf4 Enhances reprogramming efficiency Inhibits DNA MTase and induces fully reprogrammed iPS state Inhibits G9a histone MTase and increases reprogramming efficiency Inhibits DNA MTase and facilitates reprogramming of fibroblasts L-channel calcium agonist, supports reprogramming of MEF Inhibits HDAC and improves MEF reprogramming efficiency
Thiazovivin
Human fibroblasts
AMI-5
MEF
8-Bromoadenosine 30 ,50 -cyclic monophosphate (8-BrcAMP) Dasatinib PP1 iPYrazine (iPY)
Human foreskin fibroblasts
Inhibits HDAC and improves MEF reprogramming efficiency with four iPSC TFs Inhibits TGF-b receptor, enhances MEF reprogramming efficiency in the absence of c-Myc or Sox2, and enhances and accelerates reprogramming of human somatic cells Inhibits ROCK, and enhances and accelerates reprogramming of human somatic cells Inhibits protein arginine MTase, in combination with A-83-01, enables reprogramming of MEFs with Oct4 only Activates PKA and improves reprogramming efficiency
MEF MEF MEF
Src family kinase inhibitor, replaces Sox2 in MEF reprogramming Src family kinase inhibitor, replaces Sox2 in MEF reprogramming Src family kinase inhibitor, replaces Sox2 in MEF reprogramming
a
The seven small molecules combined that can fully reprogram mouse fibroblasts [42] are highlighted in italic type. Abbreviations: EZH2, enhancer of zeste homolog 2; HDAC, histone deacetylase; LSD1, lysine-specific demethylase 1; MEF, mouse embryonic fibroblast; MEK, mitogen-activated protein kinase kinase; MTase, methyltransferase; PDK1, phosphoinositide-dependent kinase-1; PKA, protein kinase A; TF, transcription factor. b
transgenic mice, the signaling pathways [e.g. the Wnt signaling, the Sonic hedgehog (SHH) signaling and the TGF-b signaling pathways] regulating various adult stem cells are beginning to be revealed. There is good reason to believe that human adult stem cells are similar, although species-specific differences might exist. This knowledge makes enhancing tissue regeneration through regulating endogenous adult stem cells an attractive strategy. Many genetic studies have demonstrated that impairing signaling pathways that are important to adult stem cell maintenance results in deletion of the adult stem cell pool [48,49]. In regenerative medicine, it is desirable to enhance the adult stem cell pool through manipulating these important signaling pathways. For example, erythropoietin infused into the brains of mice increased production of neural progenitors from neural stem cells [50]. Mildly increasing Wnt signaling levels through genetic manipulation enhanced hematopoietic stem cell function [51]. Although protein infusion to the brain and genetic manipulation are impractical in humans, regulating adult stem cells with small molecule drugs is likely to become feasible. An encouraging example is the activation of SHH signaling to stimulate hair generation in mouse skin through one topical application of a small molecule SHH agonist [52].
Overactivation of Wnt, SHH and Notch signaling is associated with tumor formation and, currently, there are many studies searching for inhibitors of these signaling pathways to explore their potential in cancer therapy. Fewer studies have reported activators (agonists) of these signaling pathways. Reasons for this bias include the overwhelming need and interest in finding drugs to eradicate cancer, and the concern relating to tumorigenic risks of the signaling pathway activators. It is unclear whether these signaling pathways are intrinsically easy to inhibit but hard to activate. The risks of tumorigenicity from small molecule drugs activating adult stem cells could be controlled by transient and local drug delivery. This can be achieved by controlled release of small molecule drugs by implanted scaffolds and injectable gels [53]. In addition, the chance of cancerous cell formation from transient stem cell activation is unclear at present. Furthermore, those patients being treated might have stem cell deficiency. Thus, in the future, the tumorigenic risk of activating adult stem cells by small molecule drugs for tissue regeneration might not be such a formidable challenge. Some small molecules regulate adult stem cells, mainly hematopoietic stem cells, neural stem cells and mesenchymal stem cells (reviewed in [54]). Tetraethylenepentamine (Gamida Cell Ltd) and 16,16-dimethylpro-staglandin E2
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Small molecules promoting iPSC generationa,b
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TABLE 3
Examples of cell fate changea,b Starting cell Genetic approaches b cells Amniotic cells Exocrine cells Fibroblasts Reviews POST SCREEN
Human fibroblasts Myelomonocytes Small molecule-induced changes Annulus fibrosus cells C2C12
Target cell
Transcription factors and/or chemicals
Macrophages Vascular endothelial cells b-cells Myoblasts Hepatocytes Neuron Cardiomyocyte Blood progenitors Neural cells Cardiomyocytes Angioblast-like progenitors Cholinergic neurons Neuron Erythroid-megakaryocytic cells, eosinophils
C/EBPa/C/EBPb ETV2 and other factors PDX1/NGN3/MAFA MyoD HNF1a/FOXA3/GATA4; HNF4a/FOXA1 (or FOXA2 or FOXA3) ASCL1/BRN2/MYT1L; or miRNAs; ASCL1/NURR1/LMX1A GATA4/MEF2C/TBX5; GATA4/BAF60C/TBX5; GATA4/HAND2/MEF2C/TBX5 OCT4 OCT4/SOX2/KLF4/C-MYC OCT4/SOX2/KLF4/C-MYC OCT4/SOX2/KLF4/C-MYC NGN2, facilitated by small molecules forskolin and dorsomorphin ASCL1, NGN2, facilitated by small molecules SB431542 and Chir99021 GATA1
Skeletal progenitor cells Multipotent mesenchymal progenitors
Reversine (MEK1/NMMII/Aurora inhibitor) Reversine (MEK1/NMMII/Aurora inhibitor) Hesperadin (Aurora inhibitor) VX-680 (Aurora inhibitor) MLN-8054 (Aurora inhibitor) PD173074/PD0325901/Chir99021/A-83-01/Parnate Butyrate (HDAC inhibitor) Trichostatin A (HDAC inhibitor) MS-275 (HDAC inhibitor) Apicidin (HDAC inhibitor) BRD7389 (inhibitor of ribosomal S6 kinases) Myoseverin (binding to microtubules) Dexamethasone (inducing C/EBPbeta)
EpiSC Oligodendrocyte precursor cells
Multipotent progenitors mESC Multipotent neural stem cells
Mouse a-cell line aTC1 Myotubes Pancreatic cells
b-cell Mononucleotide cells Hepatocytes
a
Please refer to Table S1 in the supplementary material online for references relevant to this table. Abbreviations: ASCL1, Achaete-scute homolog 1; C/EBP, CCAAT/enhancer binding protein; ETV2, ets variant 2; EZH2 (NMMII), enhancer of zeste homolog 2; FOXA3, forkhead box A3; GATA1, GATA binding protein 1; HAND2, heart and neural crest derivatives expressed; HDAC, histone deacetylase; HNF, hepatocyte nuclear factor; LMX1a, LIM homeobox transcription factor 1, alpha; MEK, mitogen-activated protein kinase kinase; MYTL1, myelin transcription factor 1-like; NGN, neurogenin; NURR1, nuclear receptor related 1 protein; PDX1, pancreatic and duodenal homeobox 1; TBX5, T-box transcription factor. b
(FT1050, Fate Therapeutics) have been studied for their activity to improve the proliferation and function of cord blood cells ex vivo.
Generating target cells by small molecule induced cell fate conversion Using genetic strategies, it is now possible to change cells directly from one cell fate (mostly fibroblasts) into another, a process called transdifferentiation (Table 3). It is evident that fibroblasts can be converted into cells from all three germ layers. Two strategies have been used: (i) forced expression or ablation of lineage-specific regulators, such as transcription factors and miRNAs (highlighted in Table 3); and (ii) timed, forced expression of iPSC-reprogramming factors, combined with lineage-specific culturing conditions [55]. Although most work on cell fate conversion was performed in vitro, endogenous exocrine cells can be transformed into b cells in vivo [56], and murine cardiac fibroblasts can be reprogrammed into cardiomyocytes by virus-mediated transcription factor expression in vivo [57]. Fibroblasts are abundant in many tissues. The ability to transform fibroblasts directly into the missing cell type important to the normal function of an organ (e.g. myocytes in skeletal muscle, cardiomyocytes in the heart, neurons in the brain and hepatocytes in the liver) will facilitate the restoration of normal function. 6
Currently, cell fate conversions are mostly achieved by genetic approaches that upregulate or inhibit genes that regulate cell fates. To eliminate safety concerns, it is hoped that small molecule drugs might be able to change the existing cells in situ into the cell types that are missing. Small molecule-induced cell fate changes have been described, although in most cases the process is de-differentiation (changing from a more differentiated state to a less differentiated state) (Table 3). With the exception of pancreatic to hepatic conversion in some pathological conditions, most chemical-induced cell fate changes have been observed in vitro. In regenerative medicine, two approaches could be adopted for small molecule drug-mediated trans-differentiation in vivo. First, small molecules controlling the expression of lineage-specific regulators can be searched for in terms of their capacity to change cell fate, which can then be tested. Second, timed treatments with iPSC reprogramming chemicals [42] could be tested for their ability to change cell fate in vivo. Although it is difficult to control the cell environment in vivo to favor cell fate conversion into a specific lineage, this might not be an issue because each organ would be the best environment for the differentiation of its resident cells. This view is supported by the observation that the in vivo reprogramming of fibroblasts into cardiomyocytes by transcription factors was more complete than
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the in vitro process [57]. However, several questions still remain: for example, how much of the in vitro work can be replicated in vivo? How tightly can this process be controlled? When a given disease is present, how can the cell environment be made favorable for cell fate conversion?
Screening for small molecules with potential use in regenerative medicine Although biochemical HTS has been widely used in the pharmaceutical industry, cell-based HTS is frequently used to screen for small molecules that could be useful in regenerative medicine. Most small molecules listed in this review were found by screening using PSCs, mesenchymal stem cells, neural stem cells and hematopoietic stem cells, which are readily available. Cell-based HTS using other types of adult stem cell has been difficult because of the limited availability of these cells. Zebrafish is a suitable system amenable to HTS for small molecules regulating adult stem cells and 16,16-dimethylpro-staglandin E2 (FT1050) was found by using this system [58]. Intestinal organoids [59] and liver organoids [60] culturing protocols have been developed that might make screening for small molecules regulating intestine and liver stem cells possible.
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inhibitor, Italfarmaco) and HT-100/halofuginone (Halo Therapeutics, LLC) for promoting muscle regeneration in patients with Duchenne muscular dystrophy. Currently, hESCs and hiPSCs have garnered the most attention for their potential in regenerative medicine, and small molecules modulating these cells are being actively investigated. Small molecules regulating adult stem cells in vivo are likely to find wide application in regenerative medicine, and HTS with organoids might help to find such small molecules. At present, little attention is being paid to the use of small molecules to enhance cell–matrix interactions. However, with interest in acellular matrices increasing, research into this area is also likely to increase. There are still many challenges in the development of chemical leads into drugs for regenerative medicine. One is that the in vitro performance of a small molecule does not necessarily predict its in vivo performance. For example, isoxazole showed promising cardiogenic activity in vitro, but had no beneficial effects in myocardial infarction models. In addition, the tumorigenic risk of activating endogenous stem cells with small molecules to enhance tissue regeneration needs to be determined. Despite these challenges and questions, small molecules and small molecule drugs are certain to have an increasingly important role in regenerative medicine.
Current status and future directions Although currently small molecule drugs meeting the definition of regenerative medicine are still under development, this situation is likely to change in the future. Several small molecule drugs in clinical trials can be regarded as regenerative medicine. These include nitric oxide-releasing gel (sponsored by China Medical University Hospital) for promoting hair regeneration in subjects with androgenic alopecia, and Givinostat (a histone deacetylase
Acknowledgement The authors thank Karen Klein for editing the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.drudis. 2013.11.011.
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