Stem cell research and clinical development in tendon repair

204 Muscles, Ligaments and Tendons Journal 2012; 2 (3): 204-211

Review article

Paola Filomeno1,Victor Dayan2,Cristina Touriño3

1 Hospital de Clínicas. Facultad de Medicina. Universidadde la República

2 Hospital de Clínicas. Facultad de Medicina. Universidadde la República, Uruguay

3 Hospital de Clinicas. Facultad de Medicina. UDELAR,Uruguay

Corresponding author:Cristina TouriñoHospital de Clínicas. Facultad de Medicina, Universidadde la República Av. Italia S/NMontevideo, Uruguaye-mail: [email protected]

Summary

Stem cells are one of the most fascinating areas in re-generative medicine today. They play a crucial role indevelopment and regeneration and are defined ascells that continuously reproduce themselves whilemaintaining the ability to differentiate into various celltypes. Stem cells are found at all developmentalstages, from embryonic stem cells (ESCs) which dif-ferentiate into all cell types, to adult stem cells (ASCs)which are responsible for tissue regeneration. Stud-ies using animal models have shown promising re-sults following cell therapy for induced injury in mus-culoskeletal system, including tendon healing, butthe results can be variable. Alternative sources forcell therapy in tendon pathology may include ESCs,ASCs (bone marrow, adipose tissue or tendon de-rived stem cells) or induced pluripotent stem cells (iP-SCs). While ethical and safety concerns currentlyforbid clinical application of ESCs and iPSCs, initialclinical trials with ASCs are promising.

Key words: cell therapy,embryonic stem cell, inducedpluripotent stem cells, adult stem cells, mesenchimalstromal cells, tendon repair.

Introduction

Stem cells have several distinct characteristics that dis-tinguish them from other cell types and are one of the

most fascinating areas in regenerative medicine today.These cells are unspecialized, self-renewing and canbe induced to differentiate into various cell lineages1,having a crucial role in the development and regener-ation of human life. Stem cells are defined as cells thatcontinuously reproduce themselves while maintainingthe ability to differentiate into various cell types. Theyare found at all developmental stages, from embryonicstem cells which differentiate into all cell types foundin the human body to adult stem cells which are re-sponsible for tissue regeneration. General opinion pos-tulates that clinical therapies based on stem cells mayhave the potential to change the treatment of degen-erative diseases and severe traumatic injuries in the“near” future. During embryogenesis, a single fertilized oocyte gives riseto a multicellular organism whose cells and tissues haveadopted differentiated characteristics or fates to performthe specified functions of each organ of the body. As em-bryos develop, cells that have acquired their particular fateproliferate, enabling tissues and organs to grow. Even af-ter an animal is fully grown many tissues and organsmaintain a process known as homeostasis, in which ascells die, either by natural death or injury, they are replen-ished. This remarkable feature has ancient origins, datingback to the most primitive animals, such as sponges andhydrozoans. Mammals seem to have lost at least someof this wonderful plasticity, however, the liver can partiallyregenerate providing that injury was not too severe, andepidermis and hair can readily repair when wounded orcut. Additionally, epidermis, hair, small intestine, andhematopoietic system are all examples of adult tissuesthat are naturally in a state of dynamic flux. Even in theabsence of injury, these structures continually give rise tonew cells, which are able to transiently divide, terminallydifferentiate and die.The fabulous ability of an embryo to diversify in all celltypes and certain adult tissues to regenerate throughoutlife is a direct result of stem cells, a natureʼs gift to multi-cellular organisms. Stem cells have both the capacity toself-renew, that is, to divide and create additional stemcells, and to differentiate along a specified molecularpathway. Embryonic stem cells are very nearly totipotent,reserving the elite privileges of choosing among most ifnot all of differentiation pathways. In contrast, stem cellsthat reside within an adult organ or tissue have more re-stricted options, often able to select a differentiation pro-gram from only a few possible pathways. A new type ofcell, which has been recently engineered, is the inducedpluripotent stem cell (iPSC). These cells represent thebridge between adult (ASCs) and embryonic stem cells(ESCs), while they are adult cells, genetic modificationrenders them with embryonic characteristics (Tab. 1).

Stem cell research and clinical development in tendon repair

Embryonic stem cells (ESCs)

Emanating from the pioneering mouse research of Mar-tin Evans2 in the 1970s and culminating with the recentsuccessful experiments with human tissue3, cells fromthe inner cell mass (ICM) of mammalian blastocystscan be maintained in tissue culture under conditionswhere they can be propagated indefinitely as pluripotentembryonic stem cells. If injected back into a recipientblastocyst, which is then carried to term in a female host,these cells can contribute to virtually all tissues of thechimeric offspring, including the germ cell compartment.To maintain cultured ESCs in their relatively undifferen-tiated pluripotent state, they must both express the in-trinsic transcription factor Oct4 and constitutively re-ceive the extrinsic signal from the cytokine LeukemiaInhibitory Factor (LIF)4.Upon LIF withdrawal, cultured ESCs spontaneously ag-gregate into embryo-like bodies, where they differentiateand spawn many cell lineages, including beating heartmuscle cells, blood islands, neurons, pigmented cells,macrophages, epithelia, and fat-producing adipocytes5.Similarly, when ESCs are injected into nude mice, theydifferentiate into multicellular masses, called teratocar-cinomas. Although the programs of gene expression in thesestructures often bear strong resemblance to the differ-entiation pathways typical of developing animals, thetriggering of these programs is chaotic, yielding a jum-bled grab bag of tissue types. These examples graphi-cally illustrate the importance of intercellular interac-tions and cellular organization in orchestratingdevelopment and embryo shape.Due to their enormous capacity to differentiate into almostevery cell type of the body and potentially replenish dam-aged tissues, ESCs have been used experimentally in di-verse animal models. They have been shown to suc-cessfully replace neural, cardiac, hepatic, hematopoietictissue among others6. Nonetheless, ethical considera-tions and safety concerns (risk of teratoma formation and

immune rejection upon transplantation) are the main lim-itations of their use in humans. First human ESCs were isolated in 1998 by Thomson etal.3 from in vitro fertilization clinics embryos. Few humanESC lines are available and there is a concern over thegenetic stability after long-term amplification in vitro7.Nowadays only 2 trials have been authorized to useESCs in humans: an US company endorsed trial for neu-rodegenerative disease which never made it to a peerreview journal and recently, the first European trial hasbeen given green light to start recruiting patients withmacular degeneration8. Both trials have short term fol-low-up (both started in 2011) and therefore potentialside effects such as teratoma formation is not estab-lished. Immunological issues (rejection) associated with the useof ESCs could be overcome by the technique known assomatic cell nuclear transfer (SCNT)9. When a nucleusfrom a differentiated somatic cell, is transplanted into anenucleated oocyte, nuclear reprogramming is initiated,leading to the generation of an entire individual, which isgenetically identical clone of the original somatic cell.Generation of pluripotent cells by SCNT has been welldocumented in mouse and other animal models7. Thiscould generate custom-made, patient-specific ESCswhich can be induced to differentiate and then trans-planted without immune rejection since they have the ge-netic material of the patient. However, this technique isvery labour-intensive. Reprogramming by nuclear trans-fer technique has not been extensively demonstrated inhumans since oocyte provision is not only a rare oppor-tunity, but also an ethical concern of the moment. Anothermethod to generate human pluripotent cells is cell fusionbetween somatic cells and human ESCs which is lead-ing to the birth of “heterokaryon”. Since the repro-grammed cells contain chromosomal materials from bothcell types and exhibit chromosomal tetraploid, it needs tobe clarified whether the differentiated progeny from thesehybrid cells are functional and their risk for neoplasic celltransformation7.


Muscles, Ligaments and Tendons Journal 2012; 2 (3): 204-211 205

Table 1 - Attributes, limitations and ethical concerns of different stem cell types for cellular therapy.

Adult stem cells (ASCs)

Adult stem cells were described 50 years ago in themouse bone marrow10,11. Hematopoietic stem cells(HSCs) have been classically defined as bone marrow de-rived cells that are capable of both self-renewal and mul-tipotential hematopoietic differentiation and give rise to allblood-cell lineages. Later, Friendenstein et al.12 foundanother cell population with stem cell-like characteristicsin the bone marrow and called them colony forming unit-fibroblast, now known as mesenchymal stromal/stemcells (MSCs). Later on, such cells were found in almostevery tissue of the body, and were broadly categorizedinto adult stem cells. While diversification of cell types is largely complete ator shortly after birth, many adult tissues undergo self-re-newal and accordingly must establish a life-long popu-lation of relatively pliable stem cells. Adult stem cells areoften relatively slow-cycling cells able to respond tospecific environmental signals and either generate newstem cells or select a particular differentiation program.When a stem cell commits to differentiate, it often firstenters a transient state of rapid proliferation. Upon ex-haustion of its proliferative potential, the cell withdrawsfrom its cycle and executes its terminal differentiationprogram13. Adult stem cells are localized to specific niches, wherethey use many of the stimulatory and inhibitory signalsused by their embryonic counterparts for selecting a spe-cific fate. ASCs have been found in the following tissues:bone marrow, liver, umbilical cord, brain, adipose tissue,gut, among others. Cells found in each of these nichesshare the same properties: self-renewal and differentia-tion capacity. In contrast to ESCs, ASCs are more re-strained to their differentiation possibilities and are favoredtowards the tissue in which they reside. Such that mus-cle stem cells differentiate mainly towards myoblasts andhematopoietic stem cells towards blood cells.Recent investigations show a regulatory role for bloodvessels in these specialized niches. Mesenchymal stro-mal/stem cells (MSCs) are located surrounding capillar-ies in a variety of tissues and have the capacity to differ-entiate into different mesodermal lineages. Angiogenicprogenitor cells have also been found in the adventitiallayer of large vessels. In the bone marrow, endothelialcells control hematopoietic stem cells release, and in thebrain, blood vessels regulate neural stem cells self-re-newal and neurogenesis. Similarly, perivascular progen-itor cells have also been found in the heart. This intimateconnection between stem cells and the vasculature con-tributes to tissue homeostasis and repair14. Knowledge of new stem and progenitor cell populationsin the body is accumulating at a rapid pace and a new eraof targeting resident stem cell populations for therapeu-tic purposes is coming into focus.

Hematopoietic Stem Cells (HSCs)

Despite its complexity, blood is probably the best under-stood developmental system. Easily accessed, bloodand bone marrow have been object of study for many

years. Direct sampling of the hematopoietic tissues inthe bone marrow presents a rather low bar for biopsy ac-quisition from living donors, especially when comparedto other systems such as heart or brain. This relativelyeasy procedure to obtain cells from primary anatomicallocation of blood cell genesis and differentiation as wellas a reliable transplantation assay and well-describedsurface markers make HSCs the best understood of alltissue stem cells. Clinically, HSCs transplantation represents the mostwidely deployed regenerative therapy, but human HSCshave only been characterized recently. Over the past 10years, increasing evidence has accumulated that het-erogeneity is a feature of HSCs proliferation, self-re-newal, and differentiation based on examination of theseproperties at a clonal level15. Recent progress in thehematopoietic field has included identification of HSCs ca-pable of long-term engraftment at the single-cell level, im-provements in ex-vivo expansion of HSCs, transdifferen-tiation of somatic cells into hematopoietic progenitors, andthe ʻcorrectionʼ of several disease-specific iPSCs usingvarious gene-targeting strategies16.The HSCs, commonly used for stem cell transplantationcan be obtained from bone marrow, peripheral blood orumbilical cord blood. Adult bone marrow, situated withinthe bone cavity, comprises three distinct stem cell pop-ulations: HSCs, mesenchymal stromal/stem cells(MSCs) and endothelial progenitor/stem cells (EPCs).The homeostasis inside the bone marrow and within theentire body is sustained by an intricate network of growthfactors and transcription factors that orchestrate theproliferation and differentiation of these multipotentstem/progenitor cells. A small proportion of cells in pe-ripheral blood are actually pluri/multipotent stem cells.These peripheral blood stem cells (PBSCs) are thoughtto be heterogeneous and could be exploited for a vari-ety of clinical applications. The exact number of distinctpopulations is unknown. It is likely that individual PBSCpopulations detected by different experimental strategiesare similar or overlapping but have been assigned dif-ferent names. Zhang et al.17 divide PBSCs into sevengroups: hematopoietic stem cells (HSCs), CD34- stemcells, CD14+ stem cells, MSCs, very small embryonic-like (VSEL) stem cells, endothelial progenitor cells(EPCs), and other stem cells. Umbilical cord blood(UCB) was initially employed in the treatment of bloodmalignancies due to its high concentration of hematolog-ical precursors and is now a non-controversial and ac-cepted source of HSCs and non-hematopoietic progen-itors cells for a variety of emerging cell therapies inclinical trials18.

Mesenchymal Stem/ Stromal Cells (MSCs)

Mesenchymal stem cells, also called mesenchymal stro-mal cells, bone marrow stromal stem cells, multipotentadult progenitor cells, mesenchymal adult stem cells ortissue stem cells, exist in almost all tissues and are a keycell source for tissue repair and regeneration. These cellsare generally thought to be resident in the perivascularcompartment of these tissues19. Recent studies have

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suggested that resident in almost all tissues are a smallnumber of dormant stem cells that can become activatedand specifically migrate to sites of tissue damage, wherethey then perform repair functions20. Isolation of MSCscan be from numerous connective tissues but is mostcommon from bone marrow. MSCs were originally iden-tified by Friedenstein et al.21 as the primary transplantablecomponent of the bone marrow microenvironment neces-sary for the maintenance of definitive hematopoiesis.The term mesenchymal stem cells has been criticized, asthere is little data demonstrating self-renewal of definitivesingle-cell-derived clonal populations from a mesenchy-mal cell source22,23. The differences between MSCs pop-ulations derived from different tissues are becoming moreapparent, presenting an additional challenge to devisinga universal definition24. However, accumulating preclinicaland clinical evidence indicates that these cells are goodcandidates for cell therapy. Under pathological condi-tions, such as tissue injury, MSCs are mobilized towardsthe site of damage. Tissue damage is usually accompa-nied by proinflammatory factors, produced by both innateand adaptive immune responses, to which MSCs areknown to respond. Indeed, recent studies have shownthat there are bidirectional interactions between MSCsand inflammatory cells, which determine the outcome ofMSC-mediated tissue repair processes. In addition, MSCsmay have potential in suppressing uncontrolled immuneresponses, providing in situ negative regulation during theinflammatory response. Although the immunomodulatorycapacity of MSCs could be use therapeutically, theremay also be unwanted effects associated with immuno-supression25.The International Society for Cellular Therapy has estab-lished minimal criteria for defining MSCs26. These basalattributes include the abilities to adhere to plastic undernormal cell culture conditions, to express a set of cell sur-face antigens (CD105, CD73, and CD90) while not ex-pressing antigens indicative of other cell lineages, and todifferentiate into adipocytes, osteoblasts, and chondrob-lasts under specific conditions. This has served to allowa basis of comparison between the results of different in-vestigators and has allowed a more focused investigationfor clinical trials. MSCs as currently defined are a phe-nomenon of in vitro culture, suggesting that extrapolatingthe function of these cells to activity in vivo must be donewith caution24.In culture, most MSCs have a spindle morphology like fi-broblasts, and can be maintained for several passageswithout significant alterations in their major properties.MSCs are multipotent and can differentiate into distinctcell types, such as chondrocytes, osteoblasts, andadipocytes27. However, recent findings show that serialpassages of MSCs in culture lead to decreased differen-tiation potential and stem cell characteristics, eventuallyinducing cellular aging which can limit the success ofcell-based therapies. Other studies indicate that in vitroaging (passage number in culture) is more importantthan in vivo aging (donor age) when considering the pro-liferation and differentiation potential of MSC28. MSCsderived from adult bone marrow can be cloned and ex-panded in vitro without loss of differentiation potential;these bone marrow-derived MSCs are the most routinely

used in studies. However, many properties of these cellsremain unknown.The mechanisms underlying tissue regeneration and im-mune modulation by therapeutic doses of MSCs also re-quire further elucidation, particularly the extent to whichthe two processes intersect. The more recent apprecia-tion that MSCs may not mediate tissue regeneration by di-rect cell replacement is also likely to redirect investigationinto more fruitful directions. The effect of MSCs can bethrough differentiation toward target cell lineage but ismore likely to involve trophic modulation by paracrineand autocrine activity; secretion major histocompatabilitycomplex (MHC) of angiogenic, chemoattractant, and an-tiapoptotic factors; and specific anti-inflammatory effectsthrough reduced T-cell activity and MHC suppression29.As a result of their extensive proliferative capacity, it ispossible to produce relatively large numbers of MSCs forpotential clinical applications from bone marrow, umbili-cal cord, or adipose tissue. Several studies have providedimportant information about the safety of MSC-basedtherapy. There are over 100 clinical trials with MSCs reg-istered on clinicaltrials.gov. While a majority of these tri-als are not completed, the available data, most of whichare derived from GvHD treatment, are not conclusive,which suggests the need for larger trials with MSCs. Interms of clinical applications, MSCs are being tested infour main areas: tissue regeneration for cartilage, bone,muscle, tendon and neuronal cells; as cell vehicles forgene therapy; enhancement of hematopoietic stem cellengraftment; and treatment of immune diseases such asgraft-versus-host disease, rheumatoid arthritis, experi-mental autoimmune encephalomyelitis, sepsis, acutepancreatitis and multiple sclerosis25. In view of the extraor-dinarily rapid and extensive use of MSCs clinically, areappraisal of the approach to the development of clini-cal protocols based on confirmed laboratory and preclin-ical observations would be timely and helpful24.

Tendon-Derived Stem Cells (TDSCs)

The TDSCs have been described in 2007 by Bi et al.30.These stem cells present in mature tendon have self-re-newal and multilineage differentiation potential. They candifferentiate into other cell types, like muscle or fat cells.These cells have been implicated as possible cause ofchronic tendinopathy because of the erroneous differen-tiation into abnormal matrix components causing fattydegeneration and calcification. These cells are still in thepreclinical experimentation stage but has great potentialfor tendon therapy in the future31,32.

Induced pluripotent stem cells (IPSCs)

The iPSCs are adult somatic cells which acquire embry-onic stem cell potential after genetic modification. Theyare similar in many aspects to natural pluripotent stemcells (like ESCs), such as expression of certain stem cellgenes and proteins, chromatic methylation patterns, dou-bling time, embryoid body formation, teratoma formation,viable chimera formation, potency and differentiability.

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The iPSCs were first generated by Shinya Yamanakaʼsteam at Kyoto University, Japan in 200633. Since the abil-ity to reprogram adult cells was demonstrated, they man-aged to identify four important transcription factors thatcould induce fibroblasts to become embryonic like-stemcells known as ̒ Induced pluripotent stem cellsʼ. Yamanakaused retroviruses to transduce mouse fibroblasts with aselection of genes that had been identified as particularlyimportant in ESCs. Eventually, four key pluripotencygenes essential for the production of pluripotent stemcells were isolated; Oct-3/4, SOX2, c-Myc, and Klf4 (Ya-manaka factors). In 2007, the same investigator managedto accomplish the same achievements using human fi-broblasts34. The success of iPSCs was confirmed by lat-ter studies from multiple groups, including the combina-tion of different factors like Nanog and Lin28. These cellspassed the most stringent examinations for gene expres-sion profile, pluripotency, self-renewal and germ layerdifferentiation both in vitro and in vivo, confirming their re-markable similarity to ESCs. The iPSCs share the sameadvantages and disadvantages of ESCs with the excep-tion of their major ethical concern since they are fromadult patients and the fact that cells may be geneticallymatched to patient. But other potential drawback is thelikely presence of inherited or accumulated mutations inthe genome from older adult cells that would predisposethem to senescence or cancer. There are currently twoadditional problems with the iPSC technology: the effi-ciency of human iPSC is substantially low (less than0.1% of fibroblasts become iPSCs) and the use of virusas a vector can result in the random integration of viralDNA into the host-cell genome7.Various groups have tested successfully the use of iPSCsfor the regeneration of diverse tissues such as neural, car-diac, hematopoietic, chondrogenic and osteogenic in dif-ferent models35-37. In order to avoid genetic manipulationof adult somatic cells, investigators have tested the pos-sibility of obtaining iPSCs only using the Yamanakaʼs fac-tors as proteins to stimulate fibroblasts38. Furthermore, inorder to avoid the main caveat of iPSCs use (which is ter-atoma formation), various groups have reported directdifferentiation of adult somatic cells such as fibroblasts to-wards neural and cardiac phenotype39. Recent studieshave shown that an ESC-enriched noncoding RNA (miR-302) induces somatic cell reprogramming to form iPSCs,suggesting its pivotal role in stem cell generation. ThismiR-302-induced somatic cell reprogramming involvesan epigenetic reprogramming mechanism similar to thenatural zygotic reprogramming process in the two- toeight-cell-stage embryos and this mechanism can be useto improve iPSC generation. For regenerative medicine,the dual function of miR-302 in both reprogramming andtumor suppression has provided us a convenient meansto control the quantity and quality of iPSCs40.

Tendon regeneration: perspectives and ethical prob-lems using ESCs and IPSs

Cell therapy and stem cell research play an important rolein orthopedic regenerative medicine today. However,many elements are required to coordinate the generation

of a functional tertiary structure in orthopedic systems, in-cluding the choice of most appropriate cell type, a vehi-cle to support cells, stimulatory and coordinating paracrinefactors, malleability to change during tissue regenerationand volume limitation. Current literature provides us withpromising results from animal research in the fields ofbone, tendon and cartilage repair. While early clinical re-sults using adult stem cells are already published forbone and cartilage repair, the data about tendon repair islimited to animal studies. There are even fewer data us-ing ESCs or iPSCs. In tendon laceration or strain injury, the local environmentis often inundated with fibrovascular and later disorgan-ized fibrous tissue. After a tendon injury, the tendon nor-mally heals through scar tissue formation, which maytake up to 1 to 2 years to mature41. During this course, thecellularity of the tendon is increased, nevertheless, the in-filtrating scar fibroblasts do appear morphologically differ-ent from native tenocytes. This lack of regeneration abil-ity of adult tendons has lead to compare the healingproperties between adult and fetal tendons42,43. Resultsfrom a fetal tendon injury model show that no abnormal-ities occur in the wound, with reconstitution of the colla-gen architecture44. Thus, ESCs may have the potential fortendon regeneration. However, until nowadays, studieshave been made only in vitro or in animal models (Tab. 2).Fetal-derived embryonic stem cell-like cells have recentlybeen evaluated for tendon and ligament repair. Chen etal.45 have demonstrated that human ESCs improve bothin vivo and in vitro tendon regeneration after stepwise dif-ferentiation from ESCs to tenocytes through a MSCstransition stage. Under in vitro mechanical stress, humanESC-derived MSCs (hESC-MSCs) differentiate and formtendon-like tissues. In animal models these cells im-proved tendon regeneration both structurally and function-ally. No teratomas were found at 4 weeks after cell implan-tation, and even at 8 weeks after ectopic implantation inSCID mice and 8 weeks in rats. Watts et al.46 studies re-vealed substantial and clinically relevant improvement inthe healing of tendon injury after intra-lesional injection ofpluripotent stem cells in a large animal model. Manystudies provide evidence for the possibility of using ESCs-derived engineered grafts to replace missing tendon tis-sue47-50. Early-stage fetal tissue has been used to developan ESC-like cell line that expresses all the markers ofESCs but without the teratogenic potential, providing abetter safety profile. Evaluation of these ESCs in a newequine enzymatic/physical defect tendonitis model indi-cates advantages to the use of ESCs compared withMSCs. There are no published data documenting theoutcome in clinical trials in animals, but anecdotal data af-ter ESC injection of flexor tendon injury in several hundredhorses used in a variety of athletic pursuits are very sup-portive of future application in humans29,51. The use of hu-man ESCs as a resource for cell therapeutic approachesis an interesting alternative, however, from a legal and eth-ical point of view, research involving human embryoniccells is highly controversial and many countries are re-viewing their legislation. Besides the ethical concerns, theuse of embryonic stem cells is problematic, as the use ofallogenic pluripotent cells involves an oncogenic potentialthat currently forbids the application in patients.



Given the concurrent social and moral dilemma regard-ing harvesting of embryonal or fetal tissues as well as thebenefits of fetal derived ESCs, the iPSCs may potentiallysolve some current concerns in stem cell therapy fornonfatal disease such as tendon injury. Thus, the iPSCscould provide the possibility of autologous therapy witheasily accessible pluripotent cells. Recently, it has alsobeen developed iPSCs lines from large animals likehorses, which comprise a major subset of experimentalmodels of tendon disease and repair52. The horse hasseveral well-known models of tendon injury, and iPSCstherapy holds considerable promise as the adult-derivedversion of fetal-derived ES-like cells. However, this fieldis still evolving because problems with incomplete repro-gramming in some iPSCs lines, including the equine iP-SCs. Besides the great potential this technique undoubt-edly represents, it also bears some essential safetyproblems that are currently far from being solved. AsESCs, these cells present a high oncogenic potential,which currently forbids its application in patients.While ethical and safety concerns currently forbid appli-cation of ESCs and iPSCs in patients53 adult stem cellsraise less ethical concerns and have proved to be saferthan pluripotent stem cells (Tab. 1). In recent years, theuse of cell therapy with ASC in equine veterinary medicinehas been intensively studied and their regenerative effecthas been described in tendon and ligament injuries54,55.ASCs therapy in tendon repair likely has more a role in co-ordinating regeneration rather than supplementing cellnumbers to bridge the void in tendon architecture29. Clin-

ical application of cultured bone marrow–derived MSCsin clinical tendonitis in racehorses has resulted in im-proved return to athletic activity in long-term studies. Ap-plication of MSCs in biologic matrices generally improvesretention of cells at target sites and may improve tendonrepair. One of the consistent findings in the use of bonemarrow–derived MSCs and adipose-derived vascular-stromal fraction cells in the equine model has been theiranti-inflammatory impact. Simple autologous productssuch as bone marrow aspirate or platelet-rich plasma(PRP) provide biological agents that seem to enhance re-pair29. In humans the available evidence from clinical trials for theuse of stem cells in tendon treatment is limited31,56. Aclinical trial testing the local application of bone marrowaspirate derived from the proximal humerus at the time ofarthroscopic rotator cuff surgery have yielded positiveresults57. Gomes et al.58 investigated the effect of therapywith bone marrow mononuclear cells extracted from theiliac crest and injected into tendon borders after fixationby transosseous stiches in patients with complete rotatorcuff tears. The results suggest that this treatment is safeand has potential to enhance tendon repair. Other stud-ies investigated the use of skin-derived tenocyte-like cellsin the treatment of lateral epindondylitis and patellartendinopathy and the results show a positive effect on ten-don healing31.Increasing numbers of experimental studies describe im-proved outcome after use of a combination of stem cellsand integrated genes to stimulate stem cell function in the

Table 2 - Animal models for studying cell-therapy in tendon repair (Modified from Nixon et al.31 & Ahmad et al.29)

BDSCs, Blood derived stem cells; BM, bone marrow; BM-MSCs, Bone marrow mesenchimal stromal cells; BMP, bone morphogeneticprotein; DMEM, Dulbecco´s minimal essential medium; HA, hyaluronic acid; ID-rats, Immunodeficient rats; long DF, long digital flexortendon; M-CSF, Macrophage Colony-Stimulating Factor; MNCs, Mononucleated Cells; PGA, polyglycolic acid; SFD, superficial digitalflexor tendon.

regenerating tendon. The principal growth factors evalu-ated include bone morphogenetic protein (BMP) 12, 13,and 14; platelet-derived growth factor subunit B (PDGF-B); basic fibroblast growth factor (bFGF); insulin-likegrowth factor 1 (IGF-1) and vascular endothelial growthfactor (VEGF). Stem cells, either primed by exposure torecombinant growth factors or containing anabolic trans-genes, seems to be an appropriate technique for en-hanced rotator cuff repair. Stem cells treated with platelet-rich plasma could also become a potential a standardtreatment. No results of clinical trials in humans havebeen published29. In summary, more studies that compare stem cells fromdifferent compartments, including the recently discov-ered tendon stem cells, are needed to determine whichstem cell population has the greatest ability to enhancetendon repair. In addition, more work will be require to de-termine the optimal combinations, timing, and cell dosing.Thus, controlled studies are required to have more evi-dence and define the role of cell therapy strategies in thestandard orthopedic care.

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Stem cell research and clinical development in tendon repair

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