seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 10, 1999: pp. 433]440Article No. scdb.1999.0330, available online at http:rrwww.idealibrary.com on

i

Regenerative biology: A millen

David L. Stocum

Regenerative biology promises to be one of the biomedicalrevolutions of the 21st century. It encompasses three researchapproaches: cell transplantation, implantation ofbioartificial tissues, and stimulation of regeneration fromresidual tissues in vivo . Multipotent and pluripotent stemcells are promising for use in cell transplantation andbioartificial tissue construction. The regeneration of sometissues has been stimulated by implanted biomaterialscaffolds. Critical research issues in regenerative biology are:( )1 how to direct the differentiation of stem or progenitor

( )cells in specific directions; 2 negating immunorejection of( )transplants and implants; 3 the ubiquity of regeneration-

( )competent cells; 4 the identification and roles of factors( )that distinguish wound repair from regeneration; and 5

bioethical considerations.

Key words: cell transplants r bioartificial tissues r regener-ation in vivo

Q1999 Academic Press

Introduction

FOR 50 YEARS, organ transplants and bionic implantshave been used successfully to replace tissues andorgans damaged by injury or disease.1 However, boththese methods have significant drawbacks. A newapproach to tissue restoration is regenerative biology,which promises to be one of the biomedical revolu-tions of the 21st century. Regenerative biology en-compasses three research directions: transplantationof cells to form new tissue in the transplant site,implantation of bioartificial tissues constructed invitro from cells seeded into a biodegradable scaffold,and stimulation of regeneration in vivo from healthy

Ž .residual tissues Figure 1 . Each of these approaches

From the Department of Biology, Indiana University-PurdueUniversity, 402 N. Blackford St., Indianapolis, IN 46202, USA

Q1999 Academic Press1084-9521r99r040433q08 $30.00r0

43

al revolution

involves basic research in cell and developmentalbiology, including research on animals with extensivepowers of regeneration, such as planaria and am-phibians, as well as immunology and polymer chem-istry. The purpose of this paper is to review currentresearch in regenerative biology and the conceptualand technical issues this research must address torealize its full potential.

Cell transplantation

Multipotent and pluripotent stem cells are potentiallycapable of forming site-specific tissue upon transplan-tation. The function of stem cells in adults is toreplenish cells lost by normal turnover or by injury.Stem cells usually divide asymetrically to produce acell which becomes part of an amplifying populationof progenitors plus a stem cell.2 Mammalian bone,muscle, epithelia, and hair all regenerate via lineage-restricted stem cells.1 Liver and pancreas containlineage-restricted stem cell populations that can beactivated under conditions of severe damage.3 Bonemarrow contains, in addition to hematopoetic stem

Ž .cells, multipotent mesenchymal stem cells MSCsthat can regenerate several mesenchymal cell pheno-types,4 ] 6 and the central nervous system harbors mul-

Ž .tipotent neural stem cells NSCs that can regenerateall neuronal and glial cell phenotypes.7] 10 Embryonic

Ž . Ž .stem cells ESCs and embryonic germ cells EGCsare pluripotent cells that can differentiate into virtu-ally every tissue of the body.11,12

Multipotent mesenchymal and neural stem cells

MSCs differentiate in vitro into osteocytes, chondro-cytes, adipocytes, fibroblasts, and skeletal muscle cellsin response to combinations of dexamethasone,

1,25-dihydroxyvitamin D3, 5-azacytidine, ampho-tericin B, or growth factors, such as BMP-2 andTGF-beta.5 They have been purified to near homo-geneity and culture conditions identified that pro-mote their differentiation specifically to fat cells, car -

3

D. L. Stocum

ran

Figure 1. Diagram relating the three research sttion, implantation of bioartificial tissues, aregeneration-competent cells.

tilage, or bone.6 MSCs function in vivo presumablyto replenish more restricted stem or progenitor cellsin downstream mesenchymal tissues, or to augmentthese cells during regeneration. Marked MSCs in-fused into X-irradiated isogenic mice populate thebone marrow and make their way into bone, carti -lage, liver, thymus, and lung parenchyma, suggestingthat MSCs serve as precursors to maintain mesenchy-mal components of a variety of tissues.5,13,14 Cells thatdifferentiate into the same phenotypes as bone mar-row MSCs in response to dexamethasone and otherbioactive factors have been isolated from the connec-tive tissues of every organ of the mouse,15,16 suggest-ing that MSCs may be ubiquitous throughout thebody.

Experiments with marked bone marrow MSCs sug-gest they might augment the satellite cells activatedto regenerate damaged muscle.17 Bone marrow cellsfrom lacZ transgenic mice injected into the regener-

ating tibialis anterior muscle of scidrbg mice ap-peared in new muscle fibers after a lag time of 2]5weeks, compared to control transgenic satellite cells,which were incorporated into new muscle within 2]5days after injection. Transgenic bone marrow cells

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tegies of regenerative biology: cell transplanta-d stimulation of regeneration in vivo by

transplanted into the marrow of scidrbg mice couldalso access regenerating muscle from the circulation.

ŽHowever, the contribution of the marrow cells pre-.sumably MSCs to the regenerated muscle was mini-

mal in both cases, so the role of these cells in muscleregeneration is still unclear.

Marrow MSCs are potentially useful as autogeneictransplants to regenerate mesenchymal tissues, par-ticularly bone, cartilage, and muscle. They are easy toisolate from small aspirates of bone marrow, expandin vitro, and transfect with exogenous genes, makingthem candidates for gene therapy protocols.5 MSCsimplanted into full-thickness defects of rabbit articu-lar cartilage restored the normal pattern of articularcartilage and bone.4,18 How closely the regeneratedcartilage matches normal articular cartilage inbiochemical composition and long-term integrity,however, is unknown. MSCs injected into the tibialisanterior muscle of mdx dystrophic mice have resulted

in a small but significant increase in muscle mass anddystrophin-positive myofibers, which are constantlyregenerating via satellite cell activity.19 Transplants ofHLA-matched donor bone marrow are being used ina clinical trial on children with osteogenesis imper-

fecta caused by mutations in the type I collagen gene,in an attempt to provide them with osteocytes thatsecrete normal collagen.5

NSCs have been identified in the walls of the brainand spinal cord.7,20 These cells were thought to re-side in the subventricular zone next to the ependyma,but experiments on adult rats in which DiI was in-jected into the ventricles, or BrdU was given in thedrinking water, have shown that the ependymal cellsare labeled first and are, therefore, the actual stemcells.21 The ependymal cells divide asymmetricallyand constitutively to form an amplifying populationin the subventricular zone which replenishes neuraland glial cells of the olfactory bulb.21,22 EpendymalNSCs are also present in humans23,24 and stem cellshave also been identified in the dentate gyrus of thehippocampus in mice25 and marmoset monkeys.26

This region of the brain is concerned with learningand memory, raising the speculation that these stemcells generate new neurons to maintain or increasememory and learning. Ependymal cells in the spinalcord of adult rats divide in response to injury, butinstead of differentiating into neurons, the transitpopulation differentiates into reactive astrocyteswhich, in combination with fibroblasts, form scartissue.21 Nevertheless, cord cells have the capacity todifferentiate into neurons and non-reactive glia. Bothbrain and cord NSCs proliferate when stimulated invitro with EGF or FGF and form neurospheres thatdifferentiate into neurons and glial cells after growthfactor withdrawal.7,10,27 The differential in vivo andin vitro responses of cord ependymal cells make animportant point: depending on their environment,stem cells can be directed into either a repair orregenerative pathway.

Two experiments illustrate the feasibility of usinghuman NSCs to regenerate central nervous tissue.First, a fraction of the neurons differentiated fromrat brain neurospheres in vitro are dopamine-produc-ing cells. Injection of these cells into the brains ofrats with experimentally-induced Parkinson’s diseasepromoted substantial recovery of the animals.9 Sec-

Ž .ond, fetal human 53]74 days post-conception NSCsmarked with a retroviral lacZ construct were trans-

Žplanted into the brain ventricles of embryonic E17-.18 or neonatal rats and tracked by the lacZ marker,

a human-specific probe to the alu repeat element,

and a human-specific antibody to glutathione-S-trans-ferase.23,24 The transplanted cells migrated into virtu-ally every region of the host brain, where they dif-ferentiated into neurons and glia appropriate to eachregion. These chimeras will be very useful in studying

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Regenerative biology

the molecular mechanisms regulating the migrationand differentiation of stem cells in the normal brain.

Pluripotent ESCs and EGCs

Pluripotent stem cells can be derived from either theŽblastoderm of early embryos embryonic stem cells,

.or ESCs , or from primordial germ cells in the germi-Žnal ridges of later embryos embryonic germ cells, or

.EGCs . These cells can differentiate randomly into awide variety of organized tissues in vitro and afterinjection into embryos.28 Their pluripotent identity ismaintained by the POU transcription factor, Oct-4,which is expressed exclusively in blastomeres and thegerm cell lineage.29 ESC cultures have been es-tablished from blastomeres of fish, chicken, and avariety of mammals.30 Recently, human ES and EGcells were established from frozen blastocysts pro-duced by fertilization in vitro11 and from primordialgerm cells of 5]9-week embryos,12 respectively. Thesecells express markers characteristic of primatepluripotent cells: stage-specific embryonic antigensŽ .SSEA-3 and 4, TRA-1-60 and 81 and alkaline phos-phatase. When injected into scid mice, or allowed todifferentiate randomly in vitro, they formed deriva-tives of all three embryonic germ layers, includinggut, kidney, neural, and epidermal epithelium, carti -lage, bone, smooth muscle, striated muscle, and em-bryonic ganglia. Human ESCs express high levels oftelomerase,11 suggesting that their replicative life spanexceeds that of somatic cells, which do not expressthe enzyme and suffer age-related replicative failure.

Human ES and EG cells are particularly valuablebecause they can be used in vitro to answer questionsabout the mechanisms of human development and toscreen drugs specifically for their effects on humanembryonic cells and tissues. They are also potentialsources of cells for tissue regeneration, as demon-strated by an experiment in which cardiomyocytes,differentiated from mouse ESCs in vitro and trans-planted into the ventricular myocardium of mdx dys-trophic mice, were stably integrated into the hostcardiac muscle.31

Bioartificial tissues

Bioartificial tissues are constructed in vitro by seedingstem or differentiated cells into a natural or artificialbiomaterial scaffold. The construct is then implantedinto the body.32 Constructs can be either open tovascularization, or microencapsulated, the cells rely -

D. L. Stocum

ing on diffusion for survival. Type I collagen alone,or in combination with other extracellular matrixŽ .ECM components, such as elastin, glycosaminogly-cans and proteoglycans, is the most widely used natu-ral biomaterial because it is ubiquitous, easy to ex-tract and mold into a variety of shapes, and isbiodegradable. Other natural biomaterials are algi-

Ž . 33nate and pig small intestine submucosa SIS . Arti -ficial biomaterials in use include calcium phosphate-based ceramics, polyurethane elastomers, polyesters,polyanhydrides, and polyphosphazenes. These mate-rials provide mainly mechanical support and adhesivesurfaces for cells, and rely on the cells themselves toprovide autocrine and paracrine signals and cues thatregulate differentiation and patterning.

Bioartificial tissues have been used to replace avariety of tissues in humans and experimental ani-mals.34 The most successful have been bioartificialskin and bone. Skin substitutes are made by seedingallogeneic dermal fibroblasts into biodegradablescaffolds of collagen or gelatin to which other ECMcomponents, such as elastin or chondroitin 6-sulphate

Ž . Žare added, or into a poly lactic acid or poly glycolic.acid mesh, then overlaying this bioartificial dermis

with a split-thickness skin graft or a layer of culturedkeratinocytes.35 Skin equivalents manufactured by

Ž .Organogenesis Cambridge, MA and Advanced Tis-Ž .sue Sciences La Jolla, CA have recently been ap-

proved for clinical use.36 As the scaffold degrades, thefibroblasts synthesize a matrix of high tensile strengthconsisting of the same molecules found in normalskin ECM: dermal type I, II, and VI collagens, elastin,tenascin, fibronectin, hyaluronate, chondroitin anddermatan sulfates, the major dermal proteoglycancore protein decorin, and mRNAs for IGF-I and II,FGF-2, and PDGF.37 The microstructure of the substi-tute skin is not completely normal, however. Hairfollicles, sebaceous glands and sweat glands are notreconstituted and the cosmetic appearance of theskin is not perfect.

Bone does not regenerate across a gap. As poten-tial substitutes for metal prostheses and bone auto-grafts, experimental bioartificial constructs consistingof ceramic scaffolds loaded with autogeneic MSCshave been tested in rats to bridge gaps made in thefemur. The MSCs differentiated into osteocytes whichformed bone within the scaffold.38,39 Ceramic scaf-

folds, however, do not resorb well, thereby limitingthe amount of new bone that can be regenerated.Therefore, more resorbable materials are now beingtested as scaffolds. For example, in vitro, C3H10T1r2cells suspended in a collagen gel formed bone in a

436

three-dimensional poly-L-lactic acid scaffold in thepresence of recombinant human BMP-2.40 In vivo,rat calvarial cells seeded onto the surface of a scaffoldconsisting of hydroxyapatite suspended in a 50:50mixture of polylactiderglycolide were induced, inthe presence of beta-glycerophosphate, to form tissueresembling cancellous bone.41

Research issues in the use of cell transplantsand bioartificial tissues: directed celldifferentiation, immunorejection, and bioethicalconsiderations

Directed cell differentiation

Autogeneic or allogeneic stem cell lines will providea theoretically unlimited source of cells for transplan-tation or bioartificial tissue construction if we canidentify the specific signals and receptors that directthe differentiation of these cells in vitro into thedesired phenotypes. This will require a deep under-standing of cell cycle regulation, the expression androle of secreted signaling factors in lineage decisions,and the expression and roles of the multiple factorsthat specify terminal cell phenotypes. These impor-tant lines of research are ongoing in cell and devel-opmental biology laboratories around the world. Aninteresting question is the extent to which the envi-ronments of adult tissues might support site-specificdifferentiation of undifferentiated multipotent orpluripotent stem cells. MSCs transplanted into de-fects in rabbit articular cartilage, either alone or in ascaffold, are reported to form histologically normalarticular cartilage,4,18 but NS, ES, and EG cells havenot been tested for their response to specific adulttissue environments.

The ability to force cell differentiation in specificdirections is also crucial for the construction of bioar-tificial tissues. It is not possible to grow new tissues ororgans to their full size and function in vitro. Thus,an alternate strategy is to make a full-size scaffoldmatching the shape of the organ or tissue and seed itin many places with small patches of undifferentiatedcells. When implanted, the scaffold and cell patcheswould become vascularized, allowing the cells to pro-liferate throughout and differentiate. To this end,

the scaffold must be both conductive, i.e. have physicalproperties and topographical features conducive tocell adhesion and migration throughout the material,and inductive, i.e. contain the appropriate biologicalinformation to promote proliferation, specific dif-

ferentiation pathways, and the ingrowth of bloodvessels. In short, the ideal scaffold should be ‘smart’,duplicating the embryonic environment that directsthe original development of the tissue. Current workis directed toward the synthesis of biomimetic scaf-folds which have desirable side chains or functionalgroups incorporated into the material directly, oradded to them through links provided by chemicalmodification of the material.42 In addition, the scaf-

Žfold material needs to be biocompatible not elicit.an inflammatory reaction or immunorejection ,

biodegradable on a schedule matching the produc-tion of the new tissue, sterilizable, and cost-effective.Once its development is underway, a vascularizedconstruct might be able to sequester additional regu-latory factors from the environment that promote thedifferentiation of organized tissue. Furthermore, asits cells proliferate, they would interact in autocrineand paracrine ways to augment and expand the arrayof differentiation-inducing and organizing factorsbuilt into the scaffold.

Immunorejection

Several strategies to promote host tolerance of trans-planted allogeneic or xenogeneic organs, cells, orbioartificial tissues are being explored. Genetic modi-

43 ] 45 Ž .fication approaches include: 1 transfecting cellsŽwith the FasL gene which encodes a protein that

.kills T-cells, thus mimicking immunopriveleged sites ;Ž .2 creating transgenic donor cells which expressmolecules that inhibit the host immune response;

Ž .and 3 transfecting host bone marrow cells with agene for the enzyme glucosyltransferase uridine 59-di-

x xphosphate galactose:beta-D-galactosyl -1,4-N-acetyl -D-Žglucosaminide alpha 1]3 galactosyltransferase alpha-

.GT for short so that the B-cells derived fromhematopoetic stem cells will express Gal alpha1-

Ž .3Galbeta-1-4GlcNAc-R alpha-Gal , a surface carbohy-drate epitope targeted by xenoreactive natural anti-

Ž .bodies XNAs . This molecular chimerism inducesperipheral tolerance by shutting down host B-cellproduction of XNAs. Potential cell biological ap-

Ž .proaches are to: 1 create multiple ES or EG celllines from which to derive differentiated cells and,therefore, a wide spectrum of MHC antigens for

Ž .tissue matching; 2 create an autogeneic ESC orEGC line by fusing a host somatic cell with an enucle-

Ž .ated egg of the same or a different species; and 3Ž .create an autogeneic ‘cybrid’ cytoplasmic hybrid by

fusion of a host somatic cell with an ES or EG

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cytoplast. The feasibility of the latter two approacheshas been demonstrated by successful mammaliancloning46 and by the demethylation of several lym-phocyte genes in a thymic lymphocyte-EGC cytoplastcybrid.47

Bioethical considerations

Bioethical concerns associated with cell transplanta-tion and bioartificial tissue construction include reli -gious and philosophical beliefs regarding the source

Žof the starting material for human cells embryo or.fetus , the possibility of transmitting pathogenic xeno-

microorganisms to humans through animal celltransplants, fears of ‘monster’ experiments, such asthe formation of human-animal cell or embryo hy-brids, and questions about the degree to whichpatients can be benefited by the current state of thescience. All of these concerns need to be addressed.

Regeneration in vivo: strategies and issues

The ability to regenerate new tissue from residualtissue in situ would eliminate the problems of im-munorejection and bioethical questions associatedwith cell transplantation and bioartificial tissue im-plants. Mammals regenerate bone, muscle, epithelia,hair, and nails via resident stem or progenitor cells,and normally regenerate liver via minimal dediffer -entiation and compensatory hyperplasia of all livercell types while they maintain their differentiatedfunctions.34,48,49

A current strategy to stimulate poorly regeneratingmammalian tissues is to bridge lesions with a conduc-tiverinductive biomaterial scaffold into which regen-eration-competent host cells migrate. Numerousbiotechnology companies are engaged in research todevelop such biomimetic scaffolds for clinical use.Skin, bone, peripheral nerve, spinal cord, tendon,blood vessels, and urinary bladder have all beenstimulated to regenerate by this method.1,34 The beststructural and functional outcomes have been withscaffolds for regenerating skin and bone, but regen-eration is neither perfect nor complete in any in-stance. Some of the most intense research is focused

on regeneration of the spinal cord. Spinal cord re-generation in paralyzed adult rats has been stimu-lated by bridging lesions with intercostal nerve sheathsŽ .which serve as conduits for regeneration embeddedin a fibrin matrix impregnated with FGF-1. The rats

D. L. Stocum

recovered the ability to move and support weight ontheir hind legs, but did not recover coordinatedlocomotion.50

Two research issues are critical for achieving bettermammalian regeneration in vivo. The first is to de-termine how many tissues of the body harbor regen-eration-competent cells and the characteristics ofthese cells. If regeneration-competent cells are pre-sent in every tissue of the body, we might then beable to activate them in situ to restore damagedtissue. If stem or progenitor cells do not exist in everytissue, then it might be possible to induce theirformation via histolysis and dedifferentiation of ma-ture cells. Urodele amphibians use this mechanism toregenerate a broad spectrum of tissues and complexstructures, including limbs, tails, upper and lowerjaws, intestine, lens, and neural retina.51 Histolysis isaccomplished through the controlled degradation ofECM by a spectrum of proteinases,52 ] 54 but themechanisms of dedifferentiation are largely un-known. Urodeles also regenerate cardiac musclethrough a process of hyperplasia that superficiallyresembles mammalian liver regeneration55 and re-generate spinal cord by the epithelial-mesenchymaltransformation of ependymal cells.56 Thus, urodelesare good vertebrate models to study for insights intohow we might enhance regeneration in mammals.

The second issue is to identify the specific stimula-tory and inhibitory factors that distinguish repairfrom regeneration. One strategy to identify thesefactors is to compare and contrast the molecularenvironments and cell characteristics of tissues thatregenerate well under one set of conditions but poorlyunder another. Three such sets of conditions aredifferences in regenerative ability at different stagesof the life cycle, species-related differences in regen-eration, and mutants that affect regeneration. Speciesdifferences have been compared using newt andmouse myotubes in culture.57 Newt myotubes cul-tured at low density respond to high serum concen-

Ž .trations 20% by synthesizing DNA, whereas C2C12mouse myotubes do not. Like other cycling cells, thenewt response is via phosphorylation of the

Ž .retinoblastoma Rb protein. The difference betweenthe mouse and newt myotube response is not relatedto any differential sensitivity to standard growth fac-

tors, since none of these mimic the serum effect andmononucleate presursors of both newt and mousemyotubes have similar sensitivity to these factors.57

Tests of serum fractions on cultured newt my-otubes have identified a DNA-stimulating fraction

438

enriched in the proteolytic enzyme thrombin. Thestimulating activity was found in the sera of all speciestested and was abolished by inhibitors of thrombinactivity.58 To test whether thrombin acts directly orindirectly on the myotubes, medium containing 1.5%fetal bovine serum was incubated for 24 h withthrombin and the thrombin was then inactivated byinhibitors. This medium stimulated DNA synthesis inmyotube nuclei, whereas, control samples in whichthrombin and inhibitor were incubated together for24 h had no activity. These results suggest thatthrombin acts on myotubes indirectly by cleaving asoluble protein which binds to a receptor on newtmyotubes that may not be present in mouse my-otubes.58 Whether this protein is fibrinogen, the nor-mal substrate for thrombin in clotting blood, or someother protein, remains to be seen. The active factorgenerated by thrombin is not sufficient to drive newtmyotube nuclei through the G2rM transition, nordoes it cause dedifferentiation of myotubes tomononucleate progenitor cells.

Recently, the MRLrlpr mouse, which has a defec-tive immune system due to a mutation in the fasgene, has been shown to close ear punch holes byregeneration of cartilage and soft tissue59. This mu-tant should prove useful in analyzing the potentialrole of the immune system and other factors ininhibiting or stimulating regeneration in mammals.

Finally, an important model of differential regen-erative ability at different stages of the life cycle isfetal versus adult mammalian skin.60 Wounded adultskin repairs by scar formation, whereas, fetal skinregenerates perfectly. This difference is correlatedwith a minimal inflammatory response in fetal woundsthat appears to be related to differences in the typesand concentrations of ECM molecules and growthfactors present in fetal and adult skin.61

Concluding statement

Many basic research, technical, and bioethical chal-lenges remain to be addressed before regenerativebiology becomes a clinical reality, but the three re-

search approaches now in place should provide uswith the means to achieve this goal. Regenerativebiology promises to reverse the potential losses inpersonal freedom, quality of life, and productivitycaused by injury or disease.

38. Ohgushi H, Goldberg VM, Caplan AI 1989 Repair of seg-

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