Mesenchymal Stem Cells: Where Can You Find Them? How Can You Use Them? 10

In the last decade, the definition of stem cell has been widely and inappropriately used because of the lack of specific markers that could allow for the isolation of the true stem cell. A stem cell, by a universally accepted definition, is a cell able to proliferate indefinitely and to maintain the mul- tipotentiality throughout its lifetime. Embryonic stem cells are true stem cells, but because of the ethical concerns they raise, they have been lately overtaken by research on stem cells isolated from adult tissues.

Several laboratories have successfully isolated stem cells from many tissues: the brain, the blood, the skin, the skeletal muscle, the cord blood, and the bone marrow and many have also attempted to translate their discoveries on stem cells into clinical practice.

Nevertheless, as of today, several plugs are still missing:

• Is there a single stem cell pool from which all other tissue-specific stem cells originate?

• Are the isolated stem cell populations real stem cells?

• Does in vitro culture alter cell biology and/or cell potential and can we use it as an evalua- tion technology?

This chapter will give a brief general intro- duction on what is known about stem cells, in particular mesenchymal stem cells (MSCs), then it will concentrate on an overview of different sources of stem cells and highlight the most up- to-date disputes in the field. Finally, it will report the most promising clinical applications of MSCs, and provide a summary of the suc- cesses and failures of the technology.

Mesenchymal Stem Cells

Embryonic stem cells represent the true expres- sion of stemness: they are able to proliferate indefinitely, to self-renew, and to differentiate toward all tissues of the adult organism.

Embryonic stem cells injected in adult mice develop into benign tumors called teratomas containing cell types deriving from all three germ layers, further demonstrating their exten- sive differentiation potential.

Ethical consider- ations and a still limited understanding of the molecular mechanisms controlling the differen- tiation pathways, however, has greatly limited research in this field and consequently the use of this very promising cell population.

Following these considerations, the attention of the scientific community has turned to adult tissues and research in the past decade has focused on the isolation, characterization, and possible use of adult stem cells. Friedenstein and coworkers

were among the first to isolate mes- enchymal cells from the bone marrow and to discover the multipotentiality of the population.

They harvested bone marrow samples from the iliac crest, plated the suspension in plastic cul- ture dishes, and observed that, after a few days, by progressive removal of the hematopoietic counterpart, adhering fibroblasts started to appear attached to the bottom of the culture plate.

Their observations led to the discovery that these cells had a fibroblastic morphology, formed distinct colonies, and when implanted in vivo were able to develop into a mature bone tissue.

Since then, studies have followed that

10

Mesenchymal Stem Cells: Where Can You Find Them?

How Can You Use Them?

Anna Derubeis, Giuseppina Pennesi, and Ranieri Cancedda

159

further characterized the bone marrow-derived MSCs (BMSCs).

BMSCs have been shown to represent 0.001% of the whole marrow, they can be isolated primarily by adhesion to plastic, but could also be concen- trated by Percoll gradient centrifugation.

BMSCs are clonogenic and multipotent and are able to differentiate into bone, cartilage, hematopoiesis supportive stroma, and adipocytes

[Figure 10.1 (see color section)]. So far, nothing surpris- ing: They reside in the bone marrow and appear to contribute to the replenishment or formation of most tissues of the skeleton. Are they true stem cells? The turning point arrived when Ferrari et al.

reported that bone marrow-derived cells engrafted in injured skeletal muscle, indicating that a population in the bone marrow could colo- nize and possibly undertake a myogenic commit- ment. Similar conclusions were reached when Gussoni et al.

transplanted beta-gal bone mar- row cells in a murine model of Duchenne’s mus- cular dystrophy.

Interestingly, few reports have shown that bone marrow cells, transplanted in mice where liver damage was induced, integrated in the parenchyma.

In one case, the integration of bone marrow cells reached such a high level that a restoration of liver function was observed.

In humans, Alison et al.

were the first to show that cells within the liver had an extrahepatic origin.

These findings changed dramatically the oth- erwise generally accepted concept that the mul- tipotentiality of a MSC was restricted to lineages derived from the same germ layer and that only stem cells residing in the tissues could be acti- vated by environmental cues, possibly when injury occurred.

BMSCs are easy to harvest from the iliac crest of healthy donors and could be expanded many- fold in vitro to increase the amount of trans- plantable cells but, most importantly, they are multipotential, therefore allowing their use in a variety of clinical situations. The possibility of using BMSCs in the treatment of disease and in particular in the reconstruction of bone defects requires that they maintain their proliferation capability after transplantation and throughout the lifetime of the individual and that this poten- tial is not lost after in vitro passaging.

Here is the first problem:

• Can MSCs be expanded in vitro indefinitely?

Do cultured cells express telomerase, an

enzyme having a major role in cell immortal- ization, as embryonic cells do?

• Is the multipotentiality maintained after in vitro expansion?

Several studies have been undertaken in this respect. Bruder et al.

showed that BMSCs exten- sively expanded in vitro undergo senescence after about 38 population doublings, although maintaining the ability to differentiate toward the osteogenic phenotype. Banfi et al.

found that BMSCs in culture undergo a drastic decrease in proliferation capacity at the 23rd population doubling. Parallel to a decrease in proliferation appears a decrease in multipoten- tiality where bone formation in vivo in the nude mouse model is dramatically impaired.

Evaluation of human Telomerase Reverse Transcriptase (hTERT) expression in cultured cells and telomere length reduction during time in culture confirmed that MSCs isolated from the bone marrow age in culture and fail to respect one of the fundamental criteria of a stem cell, the pres- ence of telomerase and the maintenance of telom- ere length

[Figure 10.2 (see color section)].

Attempts have been undertaken to optimize the culture environment and growth factor require- ments needed to implement culture expansion and maintenance of the phenotype.

In partic- ular, Bianchi et al.

and Martin et al.

have reported that the addition of fibroblast growth factor-2 (FGF2) to the culture promotes a reduc- tion in the colony number and an increase in the colony size of plated BMSCs. Furthermore, the amount of cells needed to stimulate bone forma- tion in vivo is much lower than if the cells were cultured without the factor. Bianchi et al. have gone further and postulated that FGF2 acts as a selective agent that selects a more primitive sub- population within the whole cells plated.

A great contribution to the field has come from Reyes et al.

They were able to isolate from the bone marrow mesenchymal cells with stem cell characteristics by depleting the bone mar- row suspension of glycophorin A + CD45+ cells and subsequently by plating the cells on fibronectin in low serum and at low density.

Mesodermal progenitor cells in the above-

mentioned culture conditions proliferate up to

50 cell doublings without obvious senescence,

maintaining their differentiation potential. The

maintenance of the telomere length despite sub-

sequent in vitro passaging further reinforced the

stem cell nature of the isolated population.

The above article suggests that more sophisti- cated isolation procedures and possibly the identification of specific markers could greatly contribute to the selection and subsequent isola- tion of the true stem cell from adult tissues.

Every Tissue Has Its Own Stem Cell Pool

Since Friedenstein first discovered the existence of an MSC reservoir in the bone marrow, studies have accumulated that identified stem/progeni- tor cells in tissues other than the bone marrow.

Recently, Lee et al. and others

have identi- fied and isolated stem/progenitor cells from umbilical cord blood (UCB).

It is well accepted that UCB is a source for hematopoi- etic stem cells and concentration of hemato- poietic stem cells in cord blood has been shown to be greatly higher than in the bone marrow. In addition, the hematopoietic stem cells isolated from the UCB have a higher proliferative poten- tial associated with an extended lifespan.

Lee et al. were able to isolate from UCB a population of MSCs, lineage negative and glycophorin A + that were fibroblastic in shape and size, adhered to the culture dish, and were able to proliferate extensively in vitro. Furthermore, they induced UCB-derived cells toward the osteoblastic,

chondroblastic, and adipogenic phenotype in vitro by culturing the cells in specific induc- ing media presenting a behavior similar to BMSCs.

Lee et al. attempted also to investigate a neu- roglial-like and hepatocyte-like phenotype of the isolated population and succeeded in demonstrating that UCB-derived cells changed morphology assuming a glial-like phenotype and did indeed express neuroglial markers such as glial fibrillary acidic protein and micro- tubule-associated protein 2. In addition, after culturing the cells in hepatogenic medium for 6 weeks, the cells expressed albumin and exhib- ited the ability to take up low-density lipopro- tein.

More compelling evidence was given in 2004 by Kogler et al., who isolated a CD45 −, adherent cell population from the cord blood called USSCs (unrestricted somatic stem cells) that

“without spontaneous differentiation, had the intrinsic and directable potential to develop into mesodermal, endodermal, and ectodermal cell fates.”

Kogler et al. showed for the first time the ability of cord blood-derived MSCs to exten- sively proliferate in vitro up to at least 10

cells maintaining the telomere length for at least up to 36 population doublings. USSCs formed bone when implanted on a bioceramic scaffold in critical-size defects in rats and cartilage when associated to gel foams and implanted

Figure 10.2.b The diagram shows telomere length reduction in BMSCs during time in culture (black bars). The addition of FGF2 to the culture selects

for a more primitive cell population whose telomere length is higher (gray bars). The progressive shortening of telomeres in both conditions con-

firms the lack of hTERT expression. (Redrawn from Bianchi et al., 2003

.)

subcutaneously into immunocompromised mice. Furthermore, transplantation of USSCs in the preimmune fetal sheep model resulted in a substantial number of human cardiomyocytes in both atria and ventriculi of the sheep.

In this respect, MSCs from the cord blood appear slightly different from MSCs from the bone mar- row, which engrafted predominantly in the Purkinje fiber system.

This suggests that MSCs from the UCB could be more primitive than those from the bone marrow.

MSCs with extensive multipotentiality have also been isolated from the skeletal muscle of different animal models, and still controversy exists on how many types of progenitors are in the skeletal muscle and whether the cell popula- tions isolated by different laboratories are the same or exist according to a hierarchy. The gen- eral consensus is that satellite cells are the pro- genitor cells for myocytes and myotubes of the skeletal muscle and reside quiescently within the tissue.

Nevertheless, Asakura et al.

and Wada et al.

isolated satellite cells and were able to differentiate them toward the osteogenic phe- notype in vitro, after induction with bone mor- phogenetic proteins (BMPs).

Levy et al.

were the first to show that a cell population isolated from the human skeletal muscle was alkaline phosphatase positive, and up-regulated osteocalcin expression in vitro after a 24-hour induction with 1,25-dihydrox- yvitamin D3. To this respect, the theory pro- posed by Minasi et al.

(2002) is interesting, suggesting that progenitors of the mesodermal tissues are associated with the vasculature and delivered in most tissues of the body through fetal angiogenesis. Subsequently local events of the microenvironment would trigger the exit of these stem cell pools from the quiescent phase and into active proliferation.

A possible candidate for this role is the peri- cyte. Pericytes encircle capillaries in the microvasculature of the connective tissues, the nervous tissue including the cerebral cortex, peripheral nerves, and the retina, the muscle tis- sue, and the lungs.

Doherty et al.,

for exam- ple, isolated pericytes from the bovine retina and implanted them in diffusion chambers in vivo. Harvest of the implants showed forma- tion of both bone and cartilage, suggesting an extensive multipotential capability of the cell population.

After these initial findings, the search for stem/progenitor cells in adult tissues has dra-

matically spread and several stem cell pools have been identified from a variety of tissues that possess at least the potential to undergo dif- ferentiation toward the mesenchymal lineages of the skeleton. Lee et al.

and Suva et al.

have isolated MSCs from the femoral head of patients undergoing hip replacement surgery and were able to induce differentiation of these cells into at least bone, cartilage, and fat.

Osteogenic potential of cells isolated from the periosteum has been documented since 1932 when Fell

succeeded in culturing periosteal cells and appropriately induced them to form a mineralized matrix in vitro. In 1990, Nakahara et al.

were the first to demonstrate that cells isolated from the periosteum of young chicks could undergo osteochondrogenic differentia- tion in vivo when transplanted into athymic mice. Since then, extensive research has been done on periosteal-derived stem cells until 2001 when Vacanti et al.

reported the successful replacement of a distal phalanx with tissue-engi- neered bone. They isolated cells from the perios- teum and cultured them in vitro. After the cells reached 20E6, they were loaded on a coral-based scaffold reproducing the shape of the missing phalanx and under general anesthesia delivered to the site.

To this respect, it is interesting to remember that Brittberg et al.

have developed a protocol for the reconstruction/repair of cartilage dam- age: The site of the lesion is cleaned and covered with a periosteal flap sutured to the cartilage; ex vivo expanded chondrocytes are injected in the pocket and, after a variable amount of time, the lesion gets repaired. Discussions are underway to establish whether the repair is only the result of engraftment of the chondrocytes or progeni- tor cells from the periosteum contribute to the healing.

Nevertheless, as of today, to our knowledge, no proof has been shown of the abil- ity of the cells isolated from the periosteum to undergo differentiation pathways other than the osteogenic and chondrogenic ones.

Worth mentioning is the work by De Bari

et al.

(2001) who isolated stem cells from the

synovial membrane. They demonstrated that

MSCs isolated from the synovial membrane

(hSM-MSC) under appropriate cell culture con-

ditions differentiate toward the chondrogenic,

adipogenic, and osteogenic lineage. hSM-MSCs

proliferate extensively in culture without

marked signs of senescence up to 10 passages

and injected into regenerating mouse muscle

restore the muscle fibers. In addition, hSM- MSCs administered to dystrophic muscle suc- ceed in restoring the sarcolemmal expression of dystrophin.

Far more appealing for tissue engineering pur- poses is the possibility to isolate MSCs from adi- pose tissues. Several reports have been published that show the existence of MSCs in fat.

In par- ticular, Zuk et al.

were among the first to show that MSCs isolated from lipoaspiration could dif- ferentiate into bone, cartilage, and adipose tissue in vitro. They extended their study by inducing the processed lipoaspirate toward the neuro- genic and the myogenic phenotype and showed that the cells they had isolated were indeed able to express markers characteristic of the two line- ages.

Cowan et al.

have gone further by show- ing how cells isolated from the adipose tissue, ADAS (adipose-derived adult stromal) cells, can be used in a tissue engineering approach and can be considered a valid alternative to bone marrow stromal cells. In their publication, Cowan and coworkers presented healing of a critical-size cal- varial defect in rats by the implant of polylactide- co-glycolide-coated hydroxyapatite cubes loaded with ADAS cells.

What is greatly appealing about this popula- tion is that ADAS cells present several advan- tages over BMSCs: The yield of cells is much higher from fat than from bone marrow, the proliferation rate of ADAS cells is substantially higher than in BMSCs during expansion in vitro, and they are relatively easy to harvest.

Stem Cell Therapy of Skeletal Tissues

Bone Reconstruction

The ability of BMSCs to form bone has been among the first properties to be exploited. Bone formation and repair has been evaluated first in small animal models, then in larger ones, and the successful results obtained led to the first clinical trial.

Goshima et al.

for the first time developed a protocol in which cells loaded on a bioceramic scaffold and implanted subcutaneously into immunocompromised mice allowed for the evaluation of the osteogenic potential of the transplanted cell population. Bruder et al.

and Kon et al.

have gone further and evaluated the potential of BMSCs to repair bone defects

in vivo in larger animal models. Bruder et al. in dogs and Kon et al. in sheep created a bone crit- ical-size defect in the long bones and recon- structed the missing segment by implant of appropriately designed scaffolds with and with- out adsorbed BMSCs. Two months after surgery, the filled defect was examined and evaluated and bone formation was observed. In particular, even though bone formation was evidenced in both experimental and control implants, the rate of bone formation was markedly increased in implants loaded with cells. Furthermore, the bone formed had much higher stiffness when compared with the bone formed without cells.

Similar observations were conducted by Petite et al.

who repaired critical-size segmen- tal defects with in vitro-expanded BMSCs loaded on a coral-based scaffold. Efficient repair of seg- mental defects in both small and large animal models has encouraged the initiation of clinical trials. Quarto et al.

were the first to report the repair of large segmental defects in humans using a tissue engineering approach. BMSCs from the patients were harvested from the iliac crest and expanded many-fold in vitro. The shape of the defect gave appropriate informa- tion for the design of the hydroxyapatite scaf- fold and, at the time of surgery, cells were detached from the culture dish and loaded on the bioceramic. After variable time frames, all three patients presented a repair of the defect where abundant callus formation was evident.

This approach, although highly successful, still needs optimization. For example, studies are underway to establish if total, unfractioned bone marrow could prime bone repair and how fast the repair develops when compared with in vitro-expanded BMSCs.

In addition, much research is conducted on biomaterials to increase the resorbability of the implant without losing resistance. Bioceramics composed of 100% hydroxyapatite provide high strength to loading but are not resorbed and reside in the defect for several years after callus formation. On the contrary, scaffolds mainly composed of tricalcium phosphate have a greater capacity to resorption but are too fragile to sustain the weight load. Scaffolds in which different ratios of the two biomaterials are formed are currently under investigation.

Nevertheless, research on stem cells has been

lately paralleled by research on scaffolds, both

as delivery vehicles and also as active inducers

of the goal that one wants to achieve.

Cartilage Repair

The ability of BMSCs to differentiate into carti- lage in pellet culture in vitro prompted several research laboratories to investigate the possibil- ity of using these cells to repair cartilage defects.

Lesions in cartilage tissue, particu- larly articular cartilage of the knee, are subjected to the development of osteoarthritis.

In adults, when the cartilaginous tissue is dam- aged, it undergoes a repair process that involves mostly the development of fibrocartilage where the amount of collagen type I/III greatly encom- passes the physiologic amounts of collagen type II.

The substitution of hyaline cartilage with fibrocartilage impairs the viscoelastic properties of the joint. In addition, chondrocytes are rela- tively quiescent cells mostly committed toward a mature phenotype and their proliferation potential is very limited.

Therefore, lesions in the articular cartilage rarely undergo sponta- neous healing.

In 1994, Brittberg et al.

developed a very successful protocol in which they harvest a car- tilage sample from a healthy area, dissect it, and amplify chondrocytes in vitro. The lesion, sutured with a periosteal flap is filled with the chondrocyte suspension. Although healing occurs and the repaired cartilage appears hya- line cartilage, a great limitation of the protocol is the limited proliferative capacity of the cell population. Therefore, MSCs have been evalu- ated as a valid alternative as a tissue engineer- ing tool that could substitute chondrocytes in cartilage repair processes.

BMSCs present sev- eral advantages over chondrocytes: They pro- liferate extensively in culture and are relatively easy to harvest. In addition, being multipoten- tial, they can be considered a repair tool for full- thickness cartilage defects in which both the subchondral bone and the thin cartilaginous layer are damaged.

Wakitani et al.

in 1994 implanted BMSCs pre- viously in vitro expanded in a large full-thickness defect in a weight-bearing rabbit knee. Twenty- four weeks after transplantation, both cartilage and subchondral bone repaired and the defect was completely filled. Gao et al.,

in 2002, have gone further and investigated the possibility of using different scaffolds to promote differentia- tion of BMSCs toward the two lineages: They pro- posed a two-phase composite material composed of an injectable calcium phosphate and a hyaluro-

nan derivative. In this case, BMSCs loaded on the scaffold will be prompted by the calcium phos- phate to go toward bone differentiation and by the hyaluronic acid to differentiate into cartilage tissue. They concluded that by playing on the scaffold composition, BMSCs can be redirected toward the lineage of choice and efficiently repair the full-thickness defect in the rabbit knee.

Possible disadvantages of the technology that are still under investigation are the type of cartilage that forms, and possibly the inability of BMSCs to control the differentiation stage perhaps pro- gressing toward hypertrophy.

Ligaments and Tendon: The New Era

Soft tissue injuries account for half of all the musculoskeletal injuries in the United States each year. Normal healing frequently occurs through the development of scar tissue whose elastic properties are far inferior than the origi- nal tissue.

Because tendon and ligaments have a major role in the movement of the joint, their rupture or lesion could dramatically influence the stability of the joint leading to subsequent degenerative diseases.

All of this considered, investigation has begun in the use of MSCs to engineer ligaments and tendon.

A first attempt to redirect Achille’s tendon repair was conducted by Young et al.

in 1998 where a defect was created in the rabbit Achille’s tendon. BMSCs were harvested, cul- tured in vitro, and subsequently loaded on a col- lagen gel sutured to the defect. A similar protocol was used to repair a surgically induced defect in the patellar tendon.

In both studies, healing of the defect was evident but, most importantly, when compared with the control in which the scaffold was implanted without cells, greater improvement of the mechanical stability and the elastic modulus was measured.

Awad et al.

(1999) showed that BMSC- loaded implants increased to up to 15%–40%

their structural properties, keeping in mind that

the maximum force of the tendon dramatically

decreases when the medial segment is removed

to host the implant. After Awad et al.’s investi-

gations, many have attempted to reconstruct

tendons and ligaments in vivo, whereas others

have begun to engineer tendon and/or ligaments

in vitro via the establishment of bioreactors and

the use of BMSCs subjected to cyclic strength.

Altman et al.

have developed a bioreactor sys-

tem in which BMSCs are seeded on a silk matrix scaffold and placed in a bioreactor able to apply independent and well-controlled mechanical strains. The bioreactor is able to provide nutri- ment to the construct at a controlled rate and to reproduce a multidimensional strength to the developing tendon that reminds the forces applied in development. In particular, resolu- tion of less than 0.1 mm for translational and less than 0.1 for rotational strain could be applied to the developing tissue.

Immunomodulatory Role of BMSCs

In the past few years, several laboratories have begun to investigate the immunomodulatory role of BMSCs. Different independent in vitro studies performed with human or mouse cells agree that autologous or allogeneic BMSCs strongly suppress T lymphocyte proliferation, triggered by cellular, nonspecific mitogenic stimuli, or antigenic peptide, in a dose-depend- ent way.

Immunologic restriction appears not to be involved, indicating that third-party nonhistocompatible cells can be used to sup- press the lymphocyte activation.

Studies in animal models showed that BMSCs could have a critical role in the regulation of the immune response also in an in vivo setting.

Mechanisms by which this phenomenon occurs are not yet known. Autoimmune-prone mice, who have undergone allogeneic transplant with cotransplantation of donor bone as a source of stromal cells, survived 48 weeks after transplan- tation without recurrence of any autoimmune phenomenon, suggesting that stromal cells pre- vented the occurrence of graft failure, regulating T and B cell reactions.

Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurologic outcome.

BMSCs were also found in other organs and primarily localized to the vascular structures, without any obvious adverse effects. In humans, allogeneic transplantation in osteogenesis imperfecta patients resulted in 1.5%–2.0%

engraftment of donor osteoblasts, suggesting that mesenchymal precursors present in the marrow may have a potential therapeutic role.

An immunomodulatory activity could be a char- acteristic also of stem/progenitors cells of other lineages.

Experimental evidences supporting the hypothesis that BMSCs are able to regulate the immune response have been reported, indicat- ing that BMSCs suppress the activation of the immune response by mechanisms that are still unknown. The perspective of using BMSCs in preventing or curing graft rejection is intrigu- ing, especially in the case of transplant of engi- neered tissues.

Acknowledgment. Supported by funds from the Italian Ministry of Instruction, University and Research (MIUR) and from the European and the Italian Space Agencies (ESA and ASI).

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