miRNA control of tissue repair and regeneration.

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The American Journal of Pathology, Vol. -, No. -, - 2015

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Regenerative Medicine Theme Issue

REVIEW miRNA Control of Tissue Repair and Regeneration Q13

Chandan K. Sen and Subhadip Ghatak From the Center for Regenerative Medicine and Cell-Based Therapies and the Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio Accepted for publication April 2, 2015. Address correspondence to Chandan K. Sen, Ph.D., The Ohio State University Wexner Medical Center, 473 W. 12th Ave., Columbus, OH 43210. E-mail: chandan.sen@osumc. edu.

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Tissue repair and regeneration rely on the function of miRNA, molecular silencers that enact posttranscriptional gene silencing of coding genes. Disruption of miRNA homeostasis is developmentally lethal, indicating that fetal tissue development is tightly controlled by miRNAs. Multiple critical facets of adult tissue repair are subject to control by miRNAs as well. Sources of cell pool for tissue repair and regeneration are diverse and provided by processes, including cellular dedifferentiation, transdifferentiation, and reprogramming. Each of these processes is regulated by miRNAs. Furthermore, induced pluripotency may be achieved by miRNA-based strategies independent of transcription factor manipulation. The observation that miRNA does not integrate into the genome makes miRNA-based therapeutic strategies translationally valuable. Tools to manipulate cellular and tissue miRNA levels include mimics and inhibitors that may be specifically targeted to cells of interest at the injury site. Here, we discuss the extraordinary importance of miRNAs in tissue repair and regeneration based on emergent reports and rapid advances in miRNA-based therapeutics. (Am J Pathol 2015, -: 1e13; http://dx.doi.org/10.1016/j.ajpath.2015.04.001)

Injury-responsive coding genes are recognized as a main driver of wound healing and tissue regeneration.1 After injury, tissue healing is initiated either by regeneration or repair or by a combination of both. Although robust tissue regeneration is observed in a number of lower vertebrates, including urodele amphibians and teleost fish, mammalian tissue regeneration is limited, particularly in adults.2 A cornerstone in the process of regeneration is the expression of injury-inducible coding genes at the site of tissue injury. However, the simultaneous expression of an array of injury responsive coding genes, after injury, complicates signaling networks. Post-transcriptional gene silencing (PTGS) may be viewed as a filter that is aimed at selectively advancing limited sets of injury-responsive coding genes toward protein expression to streamline the repair and regeneration process. Earlier, approximately 97% of human DNA was considered as junk because it did not encode for protein.3 However, the current literature recognizes a critical role of noncoding DNA in biology. What used to be known as junk DNA is now known to produce approximately 22nucleotide long evolutionarily conserved and functionally

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critical single-stranded miRNA molecules that specifically silence mRNA function predominantly by targeting the 30 untranslated regions of mRNA.4 miRNAs play a main role in PTGS by sequence-specific inhibition of gene expression5 that may occur at different levels such as chromatin modification and silencing, translational repression, and mRNA degradation. Thus, miRNA biology determines the biological functionality of coding genes. According to the current edition of miRbase, a central online repository for miRNAs, there are 1881 hairpin precursors and 2588 mature miRNAs enlisted for humans. This list is rapidly expanding. Because the function of miRNA determines the functional fate of mRNA, understanding the importance of miRNA in the context of tissue repair and regeneration becomes critically important. Here, we discuss the developments recorded in the current literature that underscore the rapidly unfolding importance Supported by NIH/National Institute of General Medical Sciences grants GM069589, GM077185, GM108014, and NR013898 (C.K.S.). Q1 Disclosures: None declared. This article is part of a review series on regenerative medicine.

Copyright ª 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2015.04.001

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Sen and Ghatak of miRNA and related processes in tissue regeneration and repair. Q4

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Development is the Key to Regeneration In mammals, the early in utero state has provided some of the most impressive evidence of tissue regeneration.6 Such remarkable regenerative potential is markedly blunted or lost in the postnatal period and more so during adulthood and old age. Comparison of global expression pattern of mature miRNA from two groups of mouse fetal skin has provided critical insight.7 The skin at embryonic day (E)16, when the healing is regenerative and scarless, was associated with global miRNA repression compared with the skin of E19 when the healing featured adult phenotype, including scar formation.7 Of note in this context is the observation that miRNA biogenesis is substantially blunted at E16, indicative of dampened PTGS in the fetal skin.7 Consistently, miRNA function is also globally suppressed in mouse oocytes and early embryo.8 The global suppression of miRNA during mouse oocyte-to-embryo transition is likely facilitated by the expression of highly conserved RNA binding proteins Lin28a and Lin28b that are abundant during embryogenesis. Lin28a and Lin28b repress miRNA biogenesis.9 Lin28, a putative pluripotent factor, downregulates let-7 miRNA by inducing the uridylation of let-7 precursor (preelet-7) by using a noncanonical poly (A) polymerase, TUTase4.10 These reports lead to the hypothesis that fetal tissue development is enabled by transiently silencing miRNA-dependent PTGS. Such silencing of the silencer unleashes the expression of a larger number of coding genes necessary for tissue development.11 After spinal cord injury in Wistar rats, miRNA expression is markedly subdued from day 3 after injury with gradual rebound of down-regulated miRNA at 7 days after injury.12 Suppression of miRNA expression after spinal cord injury was verified in an independent study in which downregulated miRNAs were reported up to 14 days after injury.13 The pattern of miRNA suppression after injury holds across organ systems. Partial hepatectomy is followed by down-regulation of 70% of the miRNA within 24 hours of injury. Of particular interest in this study was the observation that miRNA down-regulation was preceded by a transient upregulation of miRNA 3 hours after injury. This acute-phase response of selective miRNA up-regulation was interpreted as a response aimed at priming and commitment to liver regeneration. The subsequent genome-wide down-regulation of miRNAs was required for efficient recovery of liver cell mass to support tissue regeneration. In patients undergoing auxiliary liver transplantation, down-regulation of miRNA is associated with induced cell proliferation that supported tissue regeneration.14 Failed regeneration in a similar cohort was attributed to expression of specific miRNA which enforced cell cycle inhibition and DNA methylation.14 Taken together, these observations support the hypothesis that

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injury-induced down-regulation of miRNAs may serve as an intrinsic physiologic strategy to transiently de-silence coding genes necessary for the tissue repair and regeneration processes. In adult tissue, resident stem cells (SCs) are usually postmitotic and quiescent and are substantially different from embryonic SCs (ESCs), which are actively proliferating and differentiating.15 However, external stimuli in the form of injury may suppress tissue-specific miRNAs, which in turn may lead to alterations in adult resident SCs such that they start to differentiate, akin to their embryonic counterpart, with the objective to enact tissue repair. This is one example of how regeneration is largely inspired by mechanistic underpinning of embryo development. Some of the widespread principles and processes that drive tissue regeneration include cellular differentiation, dedifferentiation, transdifferentiation, and reprogramming.16

Dedifferentiation, Transdifferentiation, and Cellular Reprogramming Unlike lower vertebrates, mammalian tissue regeneration or repair processes rely on pre-existing stem and other cells with plasticity to replace the injured tissue.17 The sources of such cell populations are limited. In response to a strong microenvironmental cue triggered by the injury, behavior of some of the cells in close proximity of the injury site changes to accommodate the emergence of new cell types. Plasticity of surviving cells at or close to the injury site provides additional pools of cell populations that may contribute to the repair or regeneration process. With the use of a fluorescent-labeled tracking method, it is observed that terminally differentiated cells may revert back to a less differentiated and more plastic state.18 The process wherein terminally differentiated cells regress from a specialized function to a simpler less differentiated state, a more SC-like state, is referred to as dedifferentiation.19 Mammalian dedifferentiation is evident in myoblasts, renal cells, oligodendrocyte precursor cells, and even in germ cells.19 The process of dedifferentiation commonly involves re-entry of regenerating cells into the cell cycle process.20 Dedifferentiation allows the cell to proliferate again before re-differentiating, permitting a variety of cell types to be replaced at the site of injury. The process of cell reversion to a more pluripotent state is demonstrated experimentally by transferring somatic cell nuclei to eggs and fusing somatic cells with pluripotent cells.21 Nuclei of fertilized egg or the pluripotent SCs (PSCs) contain some putative reprogramming factors that are capable of erasing the memory of the differentiated somatic cell. More recently we learned that a combination of only four transcription factors (ie, Oct3/4, Sox2, Klf4, and c-Myc) is sufficient to revert differentiated somatic cells into an embryonic fate somewhat akin to that of ESCs.22 The critical importance of transcription factor networks in determining stemness and cell fate was thus recognized. The emergent field of nuclear reprograming

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miRNA and Regenerative Medicine has provided critical insight into a number of fundamental cell biology processes, including transdifferentiation or direct reprogramming. Although the two terms are often used interchangeably, there are important distinctions between the two. Transdifferentiation is a spontaneous change of cell fate that typically happens in vivo under conditions of stress or pathologic insult. In contrast, direct reprogramming is induced by defined factors such as chemical compounds, transcription factors, or other artificial engineering methods.23 In other words, direct reprogramming could be defined as transdifferentiation that can only happen under stringent artificial control conditions.24 Transdifferentiation advances the notion of dedifferentiation to one step further whereby all of the cells regress to a point from where they may differentiate to multiple specialized cell types. Transdifferentiation may also bypass the pluripotent state. With the use of defined transcription factors such as Ascl1/Brn2/Myt1l, Gata4/Mef2c/Tbx5, and Hnf4a/ Foxa, fibroblasts may be reprogrammed to neurons, cardiomyocytes, and hepatocytes, respectively.25 Such inducible transdifferentiation process bypasses the pluripotent state and is therefore often referred to as direct reprogramming.26 Under normal physiologic conditions, the transdifferentiation process seems to be rare in mammals, although a closer look is warranted. However, during the early postnatal development smooth muscle of the mouse esophagus may give rise to skeletal muscle tissue.27 In adults, dedifferentiation and transdifferentiation may occur naturally during the early phase of tissue repair. These subtle changes in cellular plasticity are driven by an orchestrated regulation of gene expression networks to reconstruct tissues in a sophisticated manner that avoids undesirable consequences such as neoplasia. Although the reappearance of developmental phenotype is reported during tissue regeneration,28 the molecular underpinnings remain largely elusive. What fills part of this void is the emergent observations that span the diverse phylogenetic tree of the animal kingdom, demonstrating that miRNAs play a key role in tissue repair and regeneration.29 Given their capacity to determine the functional fate of coding genes and therefore a large network of downstream signaling pathways,30 miRNAs are rapidly emerging as potential candidates for the therapeutic modulation of tissue repair and regeneration. In this context, it is of extraordinary importance that the turnover of miRNA within the cell determines its functional fate.

miRNA Turnover Determines Cell Fate Tissue regeneration is markedly compromised with aging, and the underlying reasons remain largely elusive.31 Lin28a improve tissue repair by suppressing let-7 biogenesis and enhancing oxidative metabolism.31 Several mature miRNAs exhibit tissue-specific expression pattern, although their primary transcripts show no important changes in expression pattern.32 Although several aspects of global miRNA biogenesis pathways are well understood,5 little is known


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311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 miRNA Biogenesis 345 346 For a detailed review of miRNA biogenesis, please refer to 34,37 347 the recent literature. Broadly, miRNA biogenesis in348 volves two sequential processing events that trim the tran349 script to yield a mature functional miRNA. Apart from the 350 known canonical and noncanonical pathways, miRNA 351 biogenesis from the loop sequence, tRNA-derived RNA 352 fragments and the miRNA arm selection have now become 353 evident. Several kilobase long pri-miRNAs along with the 354 stem loop structure are cropped into a 60- to 100-nt hairpin355 structured precursor (the pre-miRNA) by RNase III Drosha 356 357 and the DGCR8 protein that constitutes the multiprotein 358 microprocessor complex along with several auxiliary co359 factors that coordinate activity and specificity of Drosha cleavage (Figure 1). Binding of DGCR8 to the pri-miRNA ½F1 360 361 facilitates precise positioning and orientation of Drosha’s 362 catalytic center approximately 11 nt upstream of the 363 stemeloop structure. In this way a double-stranded pre364 0 miRNA with a 2-nt 3 overhang is generated. The expres365 sion and activity of Drosha and DGCR8 are subject to 366 stringent regulation by multiple factors that in turn influence 367 intracellular turnover of miRNAs. For example, DGCR8 368 369 stabilizes Drosha, whereas Drosha controls DGCR8 levels 370 by cleaving hairpins present in the DGCR8 mRNA, thereby 371 inducing its degradation. The pre-miRNA is exported to the 372

about the specific mechanisms that regulate the expression of individual miRNAs that exhibit distinctive expression patterns in certain tissues.33 Interestingly, each step of the general miRNA biogenesis pathway seems to be subject to fine-tuning to allow for exquisite control of expression of specific individual miRNAs. Studies have shown that a battery of proteins take part in the regulation of miRNA biogenesis either by interacting with the Drosha-DiGeorge syndrome critical region gene (DGCR8) microprocessor complex, Dicer, or the miRNA precursors.34 At the microprocessor level, the p68 and p72 helicases regulate the biogenesis of approximately one-third murine pri-miRNA.35 Similarly, individual miRNA processing, such as priemiR21 or priemiR-7, is greatly influenced by other proteine protein interactions such as the SMADep68 complex or SMADeSMAD nuclear interacting protein 1 and splicing factor SF2/ASF.36 Similarly, heterogeneous nuclear ribonucleoprotein A1 facilitates Drosha-mediated processing by binding to the loop regions.36 The KH-type splicing regu- Q6 latory protein enhances Drosha and Dicer processing by binding to the GGG triplet motifs in their terminal loops of the pri- and pre-miRNA.36 Although the effect of Lin28 repressor is mostly restricted to let-7 family members, the nuclear factor NF90eNF45 heterodimer impairs DGCR8 binding by interacting with the stem of pri-miRNAs in a sequence-independent way.36 Finally, editing of primiRNAs or pre-miRNAs by adenosine deaminases 1 and 2 may affect mature miRNA accumulation and influence miRNA-target specificity.36

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Figure 1 The miRNA biogenesis pathway. The pri-miRNA transcript is cleaved by the microprocessor complex Drosha-DGCR8 in the nucleus. The precursor hairpin pre-miRNA is exported from the nucleus by Exportin-5eRan-GTP. In the cytoplasm, the RNaseIII Dicer forms complex with the doublestranded RNA-binding protein TRBP and AGO-2 forms the RISC. The functional strand of the mature miRNA is loaded together with AGO-2 proteins into the RISC. The mature miRNA silences target mRNAs through mRNA cleavage, translational repression, or deadenylation, whereas the passenger strand (black) is degraded. AGO-2, argonaute-2; DGCR8, DiGeorge syndrome critical region gene 8; pre-miRNA, precursor miRNA; pri-miRNA, primary miRNA; RISC, RNA-induced silencing complex; TRBP, TAR RNA binding protein.

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cytoplasm by exportin-5 via a RAN-GTP dependent mechanism. The 2-nt 30 overhang plays a crucial role in recognizing the pre-miRNA by exportin-5, permitting the export of only correctly processed miRNA precursors into the cytoplasm. Disruption of enzymes such as Drosha or DGCR8, which are essential for the miRNA maturation, results in rapid proliferation of cancer cells.38 In the cytoplasm, pre-miRNAs are further cleaved near the terminal loop by another key RNase III enzyme called the Dicer. In mouse fetal skin, Dicer is substantially downregulated, indicating suppressed global miRNA biogenesis that is likely to prevent silencing of numerous coding genes aimed at tissue regeneration often evident at E16.7 Dicer acts as a molecular ruler that cleaves the pre-miRNA at a specific distance from the ends produced by Drosha cleavage and generating an approximate 22-nt double-stranded miRNA duplex (miR/miR*) with a 2-nt 30 overhang. Dicer, together with TAR RNA binding protein, protein kinase RNA activator, and argonaute (AGO) proteins, forms the RNAinduced silencing complex (RISC). It was initially thought that RISC incorporates one of the strands of the diced miRNA duplex with the lowest base-pairing stability at the 50 end. The other strand, originally termed miR*, is degraded on the basis of thermodynamic principles and energyindependent endonuclease-mediated selection with Ago2. Strands are present in the miR-RISC complex and are named as -5p or -3p, depending on their relative position in the hairpin sequence. Evidence shows that the target abundance stabilizes the less-dominant strand of miRNA, implying that the target transcript regulation may affect the selection of mature miRNAs. We now know that 3p-miRNA is successfully activated if the pre-miRNA is 50 -capped. In addition to arm selection, the loop region of the pre-miRNA hairpin may also be used for so-called loop-miRNA, suggesting that

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double strands generated by typical miRNA processing are not absolutely needed for the functioning of certain miRNAs. The primary function of an miRNA is to target its cognate mRNA(s), a process governed by base pairing between the seed region of miRNA with the complementary sequences within the 30 -untranslated region of the targeted mRNA. Functional repression depends on the extent of complementarity. If the binding is perfect, it leads to mRNA cleavage and degradation. If the binding is imperfect, it may cause translational repression. In animal cells, miRNAs fine-tune protein translation both by direct and indirect influences. Direct effects are exerted at multiple phases of protein translation, including repression at the initiation, prevention of ribosome assembly for the initiation of translation, repression at postinitiation steps, and inhibition of elongation or termination of translation process. Indirect translational repression by miRNA may occur at the mRNA level and is caused by the destabilization and subsequent degradation and the compartmentalization and sequestration of the target mRNAs. In this mode of action, miRNAs destabilize their target mRNAs primarily through deadenylation. The compartmentalization and sequestration processes rely on cytoplasmic foci known as processing bodies where repressed mRNAs are sequestered with enriched translational repressors.

miRNA Degradation Although miRNAs are globally stable with half-lives that may extend up to days,39 it is now clear that the absolute levels of mature miRNAs are also controlled by cis- and trans-acting factors that directly regulate stability. Such specific modifications and exonucleases may profoundly influence the lifetime of any given miRNA. Stemming from these initial studies are three areas that warrant deeper

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Figure 2

Enzymes that act on the pre-miRNA and mature miRNA to determine miRNA turnover. The pri-miRNA transcript is processed by Drosha to generate the pre-miRNA. The miRNA biogenesis depends on the balance of productive and abortive ribonucleases such as Dicer and MCPIP1, both of which act on the same pre-miRNA. Argonaute and GLD-2 act on mature miRNA for maturation and stabilization. Similarly PUP-2, XRN-2, and PNPaseold35 act on mature miRNA for degradation. MCPIP1, monocyte chemotactic protein-induced protein 1; PNPase, polynucleotide phosphorylase; pre-miRNA, precursor miRNA; pri-miRNA, primary miRNA.

investigation to further elucidate the causes and effects of altered miRNA homeostasis at the level of mature miRNA stability.40 These three areas are i) miRNA modifications, ii) relation between miRNA function and stability, and iii) targeted degradation of specific miRNA.40 On an as-needed basis, reversible muting of PTGS may ½F2 be enacted by several miRNases (Figure 2) or exoribonucleases that may degrade cellular miRNA, thereby regulating downstream functional impact. The question is how do miRNAs resist attack by ribonucleases? The silencing complex miRISC may shield its resident miRNA from cleavage by ribonucleases. In fact, the core components of silencing complexes, AGO proteins, act to enhance the abundance of miRNAs besides acting as a silencer.41 Structural biology data of small RNA and AGO suggest that AGO tightly binds to small RNA. Both 50 and 30 ends of small RNA are buried within the AGO protein.42 How the AGO protein unfolds to make the miRNA accessible for exoribonuclease degradation remains unknown.

miRNA Signature in SCs

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Several studies have consistently documented that only a subset of miRNA is expressed in SCs. Deep sequencing studies have revealed that miRNA expression in naive ESCs exhibit a different signature compared with primed epiblast SCs with only one-third of the total miRNA being differentially expressed.43 Differential expression of several clusters, including miR-290/miR-295, miR-17/miR-92, miR-302/ miR-367, was reported along with a large repetitive cluster on chromosome 2.43 Although the term induced PSCs (iPSCs) is often referred to as nearly identical to their embryoderived counterparts, miRNA expression profiling between the human ESCs and human iPSCs demonstrates an important difference in a handful of miRNAs.44 Genome-wide mapping of miRNA expressed in ESCs and PSCs generated by different reprogramming strategies such as iPSCs or nuclear transfer ESCs, recognized 34 miRNAs to be differentially regulated in reprogrammed cells compared with ESCs, among which miR-24 and miR-370 were previously reported.45


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These reports demonstrate that SCs or pluripotent cells have their own distinct signature irrespective of their origin. This observation sheds light on the intriguing potential that miRNAs may function as key drivers in regeneration.46 Loss of Dicer is embryonically lethal because they lack almost all PSCs.47 In vitro, ESCs with Dicer deletion do not proliferate.48 DGCR8 ablation also blunts proliferation of ESCs which go on to accumulate in the G1 phase of the cell cycle.49 Current observations lead to the notion that an intact miRNA biogenesis pathway is required for tissue development and raises the possibility that early steps in embryonic development require the action of specific miRNAs to properly regulate SC function. Delivery of multiple members of the miR-290 family rescued proliferation in DGCR8-deficient ESCs, although they could not restore differentiation defects.50 These miRNAs are referred to as ESC-specific cell cycle-regulating miRNAs. Interestingly, proliferation and self-renewal may be blocked in ESCs by expression of let-7 miRNA, indicating that distinct subsets of miRNAs regulate SC maintenance, proliferation, and differentiation.51 Broadly, this is consistent with findings that overall miRNA complexity increases with development. More and more miRNAs are expressed as cells differentiate into tissues and organs.52 For let-7, this is additionally interesting because its expression is regulated by control of miRNA processing and maturation.53 Mature let-7 is readily detected in more differentiated cell types, whereas the precursor accumulates in ESCs and dedifferentiating tumor cells.51 Thus, the fact that miRNAs play important role in maintaining various cellular processes may be extrapolated to cellular reprogramming.

miRNAs in Cellular Reprogramming Takahashi and Yamanaka22 reported that differentiated cells may be reprogrammed to an embryonic-like state by ectopic expression of only four factors: Oct4, Sox2, Klf4, and c-Myc (OSKM, Yamanaka factors). Because the molecular mechanisms underlying such cellular reprogramming are on their way to be comprehensively understood, a growing

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621 Figure 3 Schematic illustration depicting basic 622 regulatory network of miRNA interactions aimed at pluripotency. Ectopic expression of miR-93, miR623 106a-b, and miR-302s induces pluripotency in 624 differentiated cells. Apart from direct expression of 625 core pluripotency factors such as Oct4, Sox2, KLF4, 626 and c-Myc, miR-300a-b-c-d, miR-200c, miR-369s are 627 capable of inducing pluripotency. Overexpression of 628 miR-290 may replace c-Myc during induction of 629 pluripotency. Expression of Let-7 miRNA enacts exit 630 of cells from the cell cycle by repressing LIN28. 631 Expression of miR-21 and miR-34 prevents conver632 sion of differentiated cells to their pluripotent state. 633 634 635 number of studies point toward the importance of miRNA in production of iPSCs, the statement should be used with 636 cell reprogramming. During the early phase of OSKMextreme caution. Harui et al59 have reported that in estab637 induced iPSC generation, miR-302s and miR-17 families lished cell lines adenoviral vectors do integrate into the 638 are the most abundantly expressed human ESC miRNAs.54 genome. Thus, it may not be justified to extrapolate these 639 frequencies to the in vivo situation.60 A detailed comparison Disruption of global miRNA biogenesis by ablation of 640 Drosha, Dicer, or Ago2 substantially reduces OSKMof nonintegrating nonviral methods for reprogramming 641 54 induced iPSC colonies in mouse embryonic fibroblasts. human somatic cells to iPSCs is provided in Table 1. ½T1 642 Compared with these methods, miRNA-based strategies to Introduction of several members of miR-290 cluster may 643 generate iPSCs without involving genomic integration is a enhance the efficiency of OSK-induced cell reprogramming, 644 much safer choice and therefore is of extraordinary theracomparable with that achieved by OSKM, demonstrating 645 646 ½F3 that miR-290s may substitute c-Myc (Figure 3). In this peutic value (Table 1).57,61e70 647 context, c-Myc binds to the promoter region of the miR648 290/miR-295 cluster.55 Similar studies with ectopic 649 Is miR-29 a Master Regulator of Tissue expression of miR-93, miR-106b, and miR-302 cluster were 650 Q8 successful in enhancing cell reprogramming. Regeneration? 651 In the absence of OSK reprogramming factors, delivery of 652 miR-302a, miR-302b, miR-302c, or miR-302d alone fails to In the mammalian system, the extracellular matrix (ECM) 653 56 induce the expression of epithelial markers. Two indeplays a critical role in tissue regeneration. The ECM de654 pendent groups have now demonstrated that lentiviral termines cell behavior by manipulating cell shape, prolif655 expression of miR-302/miR-367 cluster and a cocktail of eration, migration, differentiation, and death.71 Scarless 656 miRNAs (miR-200c, miR-302a-d, miR-369-3p, and miRregenerative healing is an intrinsic property of the fetal 657 658 369-5p) may reprogram human fibroblasts to iPSCs effiskin.72 Major differences in the ECM of the fetal and the 57 659 ciently. The cells that re-expressed human ESC factors that adult skin account for the sharp contrast in healing pheno660 feature global gene expression analogous to human ESCs type between the fetus and adult skin.73 Fetal skin ECM 661 were named miRNA-iPSCs. This constitutes the first proof of exhibits predominance of type III collagen, elevated hyal662 principle to demonstrate that iPSCs may be obtained with uronic acid, and high ratio of matrix metalloproteinase to 663 miRNAs without the need for genomic integration of foreign tissue-derived inhibitor that facilitates cell movements, 664 DNA. Genomic integration increases the chances of recipient turnover of ECM modulators, and tissue remodeling.72,74 665 chromosomal locus modification after integration, thereby One of the hallmarks of fetal skin development is the 666 raising the possibility of making the recipient genomic loci lower abundance of miRNAs caused by mechanisms that 667 unstable.58 Although adenovirus-derived vectors and Sendai are aimed at silencing PTGS during tissue development, 668 virus were proclaimed as safe nonintegrating methods for enabling expression of otherwise silenced coding genes that 669 670 671 Table 1 Comparison of Nonintegrating Nonviral Methods for the Human Somatic Cell Reprogramming to iPSCs 672 Methods Time, days Efficiency, % Success, % Pros Cons References 673 miRNA 20 0.002 60e70 Zero footprint Validated for only one cell type 57,61,62 674 mRNA 20e25 0.6e4.4 20e30 Zero footprint Cost, technically challenging, labor intensive 61e64 675 Protein 56 0.001 NA Zero footprint Technically challenging, reprograming time 61,64e66 676 Episomal 30 0.0006e0.02 80e90 Zero footprint Low efficiency 61,62,67,68 677 Minicircles 15 0.005 NA Zero footprint Validated for only one cell type 61,64,68,69 678 Piggybac 14e28 0.02 NA Zero footprint No data show excision of transposon from 61,70 679 iPSC, licensing/patent issue 680 681 iPSC, induced pluripotent stem cell; NA, Not available. 682

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drive skin development. Of such miRNAs that are silenced during fetal development, low abundance of miR-29 was reported in skin across several species.7,75,76 In humans, the miR-29 family consists of four highly conserved and closely related transcripts: hsa-miR-29a and hsa-miR-29b-1 from chromosome 7 and hsa-miR-29b-2 and hsa-miR-29c from chromosome 1. The members of miR-29 family share sequence homology at nucleotide positions 2 to 7, the seed region responsible for determination of the fate of the coding genes. PTGS of multiple ECM proteins by the miR29a-c family is one notable example of a single miRNA family that regulates a large set of functionally convergent genes such as different collagen isoforms, laminin 1, fibrilin 1, elastin, matrix metalloproteinase 2, hyaluronic acid, chondroitin sulfate, and integrin 1.77 In silico analyses have revealed that the 30 -untranslated region of the 20 collagen genes have predicted conserved binding sites for the miR29a-c family.78 This property is unique to the miR-29 family because the predicted binding sites of the collagen genes are not attributed to any sequence homology in their 30 -untranslated region.78 In adults, members of the miR-29 family cause suppression of ECM genes, resulting in tissue repair with a scar. Dysregulation of ECM caused by lower miR-29 was implicated in the development of fibrosis in several organs, including the heart,77 kidney,79 lung,80 and liver.81 Furthermore, miR-29 influences tissue differentiation and senescence.82 Elevated expression of miR-29 may attenuate the inhibitory action of transforming growth factor-b on myogenesis by targeting histone deacetylase 4, a key inhibitor of muscle differentiation.83 In adults, the elevated expression of miR-29 suppress the expression of insulin-like growth factor-1, p85a, and B-myb that lead to induction of senescence of muscle progenitor cells in vivo, thereby contributing to the development of muscle atrophy and sarcopenia.84 Suppression of miR-29 is a productive strategy to accelerate skeletal myogenesis by targeting its repressor YY1.42 In bones, miR-29 regulates osteoblast differentiation by targeting antiosteogenic factors and ECM proteins85 and Wnt signaling antagonists.86

influencing targets at several levels in a regulatory network to deliver robust biological output. To enable this, a thorough understanding of miRNAemRNA interactions is necessary. Developing hypotheses aimed at understanding the intricate regulatory networks with the help of in silico tools (eg, Targetscan, PicTar, mi-RANDA) and validating such hypotheses experimentally represent powerful approaches. High-throughput methods such as argonaute highthroughput sequencing of RNA isolated by cross-linking immunoprecipitation allow the study of actual miRNAe mRNA interactions in any specific biological context. The ability to properly evaluate the effect of an miRNA on a specific biological pathway will pave the way for the identification of appropriate candidates for therapeutic interventions. Because tissue regeneration is a multifaceted process, miRNA therapies must consider the differential requirements of the developmental program through which participating cells will temporally progress. Appropriate dosing and strategic targeting of specific cell populations are necessary to obtain optimal functional outcome. Because the incorporation of miRNA in the RISC complex is random in mammalian cells, any alteration in the level of a particular miRNA may also affect the activity of other miRNA by competing for incorporation into the miRISC. Cancer is associated with dysregulation of miRNA. Thus, any alteration in the miRNA pool, if not properly targeted to the tissue of interest, poses the risk of neoplastic outcome. Because there is clear overlap between key genes that control pluripotency and differentiation with those genes that are integral to cancer,90 it is necessary to meticulously design miRNA-based cell reprogramming strategies for the purposes of tissue repair and regeneration. Challenges related to intelligent target selection coupled with precision in dosing and spatiotemporal modulation of miRNA targets must be addressed. There are several methods to deliver miRNA to cells, including direct injection and viral and nonviral-based methods (Figure 4). ½F4

Cell Reprogramming Using miRNA

Direct injection is one of the simplest methods to deliver miRNA and antagomiRNAs. The effect is typically shortlived because the oligonucleotides are rapidly degraded by nucleases in serum and cleared by the kidneys for their inability to bind to plasma proteins. However, a single bolus intravenous injection of locked nucleic acideanti-miRNA may be stable and hence productive for several weeks.91

Approaches and Caution Synthetic locked nucleic acids conjugate oligonucleotides are useful to modify the cellular levels of any particular miRNA.87 Furthermore, overexpression at the level of primiRNA or pre-miRNA with some flanking sequence may be more biologically relevant than simply using the mature miRNA sequence because the transcript may interact with the endogenous cellular machinery, ensuring correct processing and final functionality.88 A key challenge in the modulation of miRNA signaling for tissue regeneration is the selection of the right therapeutic candidates. An appropriately chosen miRNA may modulate numerous functionally convergent coding genes.89 The miRNA is capable of


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Direct Injection

Viral Delivery Virus-based miRNA delivery to cells is one of the most widely used techniques in vitro.92 The most commonly used are adenoviral, lentiviral, and retroviral vectors. Although the viral vectors ensure long-term expression of the miRNA of interest (or control of expression with inducible promoters), this approach is not friendly for clinical translation because of

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Figure 4

A comparative analysis of current approaches. Although direct injection and delivery with the use of viral vectors is widely used in in vivo and in vitro research for deciphering molecular mechanisms and pathway validation, many disadvantages limit their use in translational research for therapeutic intervention. Lipid nanoparticles are comparatively safe, but they are rapidly eliminated by the reticuloendothelial system. Nanoelectroporation offers precise delivery, better transfection efficiency, and less cell mortality.

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miRNA and Regenerative Medicine Table 2

Key Control Modules in Cell Engineering Toolbox

Events

Rationality

Cell proliferation Cell death Cell migration

To ensure their survival on implantation To ensure their survival on implantation To redirect cellular movement toward specific signals and sites in the body where the cells should execute their action To reprogram cellecell, small moleculecell, and biologicecell communication For on-demand production and secretion of small molecules and biologics by engineered cells, extending beyond those molecules that a cell naturally makes For quantitative control of therapeutic cellular responses

Cell communication Cell secretions Cell response

their inherent toxicity and immunogenicity concerns.93 Adeno-associated viral vectors are promising for in vivo delivery of miRNAs because of their observed lower levels of toxicity and seemingly limited off-target effects.

Lipid Nanoparticles In the biology of tissue repair and regeneration, controlled release of bioactive reagents such as growth/differentiation factors or interfering RNA is critical to directing cell fate. These bioactive reagents may be loaded in lipid nanoparticles and supported by scaffolds for sustained local delivery.94 Lipid nanoparticles, being a nonviral vector, provide more flexibility in formulation design for improved uptake by the cells. In addition, by incorporating targeting ligands, it is possible to specify the route of vector administration and enhance the delivery to specific tissues or cells. Despite having several advantages, one of the main limitations of this approach is the absence of a three-dimensional mechanical support component frequently required to promote tissue repair and regeneration.94

Nano-electroporation Nanochannel electroporation may be used as a potential method to deliver precise amounts of miRNA into living cells. These devices are made of microchannels connected by nanochannels. With the use of an optical tweezer the cell to be transfected is positioned in one microchannel, and the miRNA of interest may be placed in the second microchannel.95 An intense electric field is produced by delivering a voltage pulse between the microchannels over a small area on the cell membrane, allowing a precise amount of oligonucleotide to be electrophoretically driven through the nanochannel, the cell membrane, and into the cell cytoplasm without affecting cell viability.95 Dose control is achieved by adjusting the duration and number of pulses. Although there is no reported in vivo evidence yet, such a nanochannel electroporation device has the potential to support tissue repair and regeneration in vivo. These highly sophisticated miRNA delivery methods cannot only deliver miRNA to cells of interest but can also be used for preparing cells with a desired miRNA payload for efficient cell based therapies.


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miRNA in Cell-Based Therapies Live cells may be used to package and deliver miRNAs of interest to tissue repair and regeneration. In this approach, paracrine effects of such implanted cells may guide tissue repair and regeneration. The lifetime of such cells can be controlled by natural and engineered circuits such as introduction of death signaling pathways96 or multiple auxotrophies (metabolic dependencies). The key control modules in the cell engineering toolbox is provided in Table 2. In cellbased therapies, the notion of paracrine effects that drive repair and regeneration is well established. For example, the beneficial activities of mesenchymal SCs are mediated by the secretion of pro-regenerative and anti-inflammatory growth factors in cardiac repair97 or graft-versus-host disease.98 miRNAs may be shuttled between cells packaged in small membrane-bound extracellular vesicles (EVs) (eg, exosomes, shedding vesicles, apoptotic bodies) that can then amend gene expression of their target cells.99 Thus, as a natural extension we hypothesize that cells could be used to deliver miRNAs to promote tissue regeneration. Alternatively, these EVs could be isolated and used to deliver a proregenerative miRNA payload. There is some interest in the use of EVs for miRNA delivery. Overexpression of miR146a in human embryonic kidney 293T cells elevated the miRNA level both in the transfected cells and EVs released from the cells.100 This approach of using cells as delivery vehicles is related to previous work that used genetically engineered MSCs to deliver proteins and to modulate in vivo tissue response.101 An advantage, particularly when using MSCs for this strategy, is that they naturally home to sites of disease or injury; therefore, they may provide a natural targeting mechanism to deliver miRNAs to the site of interest.102 MSCs transfected with mimics of miR-124 and miR-145 have successfully delivered these miRNAs to glioma cells. MSCs loaded with miRNAs of interest are able to change gene expression and cellular behavior in vivo.103 The importance of EVs in explaining such observations remain to be understood.

Tissue Engineering The ease by which the miRNAs may be overexpressed or repressed with sharp molecular tools renders them highly

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Sen and Ghatak suitable for tissue reconstruction from SCs. In some instances miRNA have overcome the need for growth factors. Currently, miRNAs are widely used in the tissue engineering of cartilage, bone, and skeletal muscle. Repairing the injured cartilage is challenging, but it holds great promise for the treatment of osteoarthritis and spinal disk herniation. Although repairing major cartilage injury by using biomaterial implant and MSCs has proven to be highly successful, the phenotype of SC-derived chondrocytes is highly unstable compared with their endogenous counterpart. Under in vivo conditions, the artificial implant undergoes terminal differentiation, followed by ossification.104 Addition of miR-133 prevents such unwanted ossification.105 Furthermore, addition of miR-675 and miR-221 promotes expression of collagen II and proliferation of mature chondrocytes. In bone engineering, seeding MSCs with miR-148b mimics and miR-489 inhibitor to the osteoinductive scaffold results in increased matrix deposition and calcification.106 Application of miR-1 and miR-206 improve the differentiation potential of human satellite cells that play an important role in the tissue engineering of the skeletal muscle.107 Biomaterial scaffolds play a key role in scar formation associated with wound healing. These ECM-derived scaffolds, in addition to serving as physical support to promote tissue organization, resist aggressive wound contraction108 and scar tissue formation.109 These scaffolds also serve as vehicles to deliver bioactive substances such as cytokines,110 growth factors,109 living cells,111 and vectors for gene therapy. Attenuated levels of collagen type I and collagen type III mRNA expression were observed when primary fibroblasts were cultured with scaffolds doped with miR-29b.112 Application of this scaffold to full-thickness wounds in vivo minimized wound contraction, improved collagen type III/I ratio, and led to a dose-dependent (0.5 versus 5 mg) increased ratio of metalloproteinase-8 to tissue inhibitor of metalloproteinase-1.112 Thus, a potential of combining exogenous miRNAs with collagen scaffolds to improve extracellular matrix remodeling after injury is evident.

Conclusion With cues from tissue development process, it is now evident that miRNAs play a critical role in tissue repair and regeneration. Such influence is executed either via endogenous repair mechanisms or by directing the activity of implanted cells at the injury site. Changes in cellular phenotype as evident during dedifferentiation, transdifferentiation, or cellular reprogramming are directly linked to miRNA function within the cell. miRNA-based approaches have opened novel avenues to produce substantial amounts of iPSC clones without the need for genomic integration. Albeit in its infancy, several lines of critical evidence have shown the promise of miRNA-based tissue reparative and regenerative therapies, including tissue engineering applications. Compared with contemporary methods of ectopic gene expression currently used in regenerative

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medicine, strategies to transiently manipulate cellular miRNA by using targeted mimics and inhibitors hold substantial promise. However, off-target effects of therapeutic miRNA remain a relevant concern. Parallel advances in novel delivery technologies such as lipid nanoparticles and nanochannel electroporation are beginning to address some of the key stability and toxicity issues, thus making miRNA-based therapies more translationally relevant. Currently, there are 152 open clinical studies on miRNAs worldwide (Clinical Trails.gov, https://clinicaltrials.gov/ct2/results?termZmiRNAþandþ Tissueþrepair, last accessed March 30, 2015). Of these, six trials are on miRNA and tissue repair, of which three are based in the United States. The race to perfect miRNA-based therapies is one of high stakes and has the clear potential to transform reparative and regenerative medicine.

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Drug delivery strategies to control macrophages for tissue repair and regeneration.

Initiation and early control of tissue regeneration - bone healing as a model system for tissue regeneration.

The benefits of oxidative stress for tissue repair and regeneration.

Inflammation and metabolism in tissue repair and regeneration.

Monocyte and macrophage plasticity in tissue repair and regeneration.

Affinity-based drug delivery systems for tissue repair and regeneration.

Fibroblast growth factors as tissue repair and regeneration therapeutics.

Modulation of tissue repair by regeneration enhancer elements.

regeneration.

Repair and Regeneration.

The cellular and molecular mechanisms of tissue repair and regeneration as revealed by studies in Xenopus.

Skin Regeneration, Repair, and Reconstruction.

Nanoscale Surface Modifications of Medical Implants for Cartilage Tissue Repair and Regeneration.

chorion allografts: therapeutic potential for soft tissue repair and regeneration.

chorion composite grafts: Implications for wound repair and soft tissue regeneration.

or Regeneration.

Tissue engineering-based therapeutic strategies for vocal fold repair and regeneration.

regeneration.

Bone tissue engineering and regeneration.

Striated muscle function, regeneration, and repair.

Regenerative endodontics: regeneration or repair?

Cardiac regeneration: epicardial mediated repair.

Telocytes in cardiac regeneration and repair.

Promoting regeneration and repair in Multiple Sclerosis.