Satellite – Non-Embryonic Stem Cells

Plasticity of marrow-derived stem cells

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Abstract

Many exciting discoveries reported over the past 3 years have caused us to expand the paradigm for understanding somatic stem cell plasticity. Within adult organs, there are not only specific stem cells that are capable of producing functional cells of one organ system, but also cells with the flexibility to differentiate into multiple other cell types. In the bone marrow, for example, in addition to hematopoietic stem cells and supportive stromal cells, there are cells with the potential to differentiate into mature cells of the heart, liver, kidney, lungs, GI tract, skin, bone, muscle, cartilage, fat, endothelium and brain. A subpopulation of cells in the brain can differentiate into all of the major cell types in the brain and also into hematopoietic and skeletal muscle cells. In this brief overview, several of these recent findings are summarized.

Introduction

The two defining characteristics of stem cells are their capability for extensive self-renewal and their potential to differentiate into at least one, and usually more, mature cell types. In any discourse on stem cells, it is important to indicate the type of stem cell and also its source. For example, a hematopoietic stem cell (HSC) is capable of differentiation into all of the mature peripheral blood cell types. Until recently, HSC were thought to reside exclusively in the bone marrow, with occasional ‘leaks’ of these cells into the peripheral blood. However, recently cells with HSC potential have been found to reside in ‘nonhematopoietic’ organs as well, including muscle. Although the concept of somatic stem cell plasticity, which suggests that stem cells can have a wide range of differentiated progeny had been suggested previously, a spate of reports over the past 3 years has brought wide attention to the concept that somatic cells in post-natal animals are highly plastic (Table 1). In this brief review, I focus on the plasticity of marrow-derived cells. In addition to the reports covered, other recent studies have also shown that there is a higher than expected plasticity of cells derived from the brain, fat, muscle and skin as well (Table 1).

Table1 Reports of adult stem cell plasticity

A spate of studies on somatic stem cell plasticity

In order to demonstrate somatic stem cell plasticity, it is necessary to (1) identify the differentiated cell as being donor derived; (2) to show that the cell is phenotypically like the resident cells of the organ; and (3) to prove that the cell is functional. Multiple approaches have been taken to identify a cell as being donor-derived. Donor cells can be distinguished from those of the recipient using male cells transplanted into female recipients or transgenic donor cells into wild-type recipients, for which the markers are the Y chromosome or the transgene, respectively. In addition, retroviral infection of donor cells can be used to ‘mark’ these cells. Phenotypic markers of differentiated cells are usually assessed by immunohistochemistry or immunofluorescence for organ-specific proteins or in situ hybridization for cell-type specific RNA expression. A further refinement on the transgenic and retroviral approaches is that the transgene can be expressed on a cell-type specific promoter so that its expression not only marks the cells as being donor-derived, but also indicates that this gene is being expressed in a cell-type specific manner. Functional assays of individual donor-derived cells have been more difficult to perform. The studies that have most successfully demonstrated functionality of the donor-derived cells have used recipient animals that are mutant for normal cell function in the target organs so that the transplanted cells restore function to the recipients.

Bone marrow subpopulations

Hematopoietic cells

Within the bone marrow, there is a veritable factory for the production of blood. Populations range from fully differentiated erythrocytes, white blood cells and platelets to the most immature cells. The existing paradigm has been that the most immature cells in the marrow are the hematopoietic stem cells (HSC) which can both self-renew and differentiate into all of the different types of hematopoietic cells. Recent data, however, suggest that there are cells in the marrow with the ability to differentiate not only into blood cells, but also into multiple other cell types throughout the body. The phenotype of these highly plastic cells in the marrow is not yet known. However, as data presented in this review show, the same subpopulations that are capable of differentiation into hematopoietic cells also have this plasticity. In humans, the HSC population is CD34+CD38 and in mice, multiple different methods have been used to enrich for HSC including CD34+lin,1 linHoechstloRhodaminelo,2 side population cells,3 linSca+Kit+Thy1lo,4 as well as others. Our widening paradigm must take into account the possibility that cells that are already partially committed to hematopoiesis may be able to be reprogrammed to differentiate into other cells types and also the possibility that some of the subpopulations of marrow cells that are enriched for cells with HSC activity may actually contain cells that are less mature than HSC and are not yet ‘committed’ to the hematopoietic lineage. Rather, they still maintain the ability to differentiate into multiple lineages.

Stromal cells

The microenvironment of the hematopoietic cells is comprised of stromal cells, a diverse population consisting of fibroblasts, smooth muscle cells, endothelial cells and others. These cells not only provide a scaffold to the developing stem and progenitor cells, but also produce extracellular matrix components and soluble proteins. Within the stromal cell population are mesenchymal stem cells (MSCs), which are capable of self-renewal, as well as differentiation into many ‘mesenchymal-derived’ tissues. The common features among the cells referred to by different investigators as MSCs is that they grow as adherent cells in culture and have the capacity to differentiate into osteoblasts, chondroblasts and adipocytes when exposed to the appropriate stimuli in vivo and in vitro.56789 Transplantation of whole bone marrow including MSCs, and hematopoietic stem cells has been used in clinical trials to treat osteogenesis imperfecta, a genetic disease affecting the bones of affected children.10 In three of three children transplanted with normal bone marrow cells including mesenchymal stem cells from allogeneic matched siblings, there was a significant decrease in the number of fractures in the first 6 months following the transplant. These promising results suggest that bone marrow transplantation may be used to correct a variety of inherited and acquired bone disorders, including those as common as osteoporosis.

Recently, a population of highly plastic adult-derived marrow cells was characterized. These cells, present in the marrow of mice, rats and humans, grow as an adherent layer in culture and can be cultured indefinitely in a relatively nutrient poor medium. Even after over 1 year of growth in vitro, these cells maintain the ability to differentiate in vitro and in vivo into multiple cell types.11 Under different culture conditions, the cells differentiate into uniform populations of myocytes, osteoblasts, chondrocytes, adipocytes or endothelial cells, all of which comprise mesenchymal tissues. The investigators have referred to these cells as mesodermal progenitor cells (MPC) or multipotent adult progenitor cells. Potential clinical uses of MPC expanded in vitro include autologous transplantation into growing or healing tissues or use as a stromal support layer for hematopoietic cells. MPC also potentially could be used for allogeneic transplantation as they express neither HLA-Dr nor HLA1 and therefore may elude immune rejection. Also, MPC can be infected with retroviral vectors and could be used in gene therapy approaches.

Bone marrow to skeletal muscle

Several studies have demonstrated that marrow-derived cells can differentiate into skeletal muscle cells. First, Ferrari et al12 used direct inject of marrow-derived cells into damaged muscle to induce differentiation of marrow derived cells into skeletal muscle myocytes. Specifically, they used bone marrow cells from transgenic animals that express the enzyme ß-galactosidase on the myosin light chain 3F promoter, which is expressed only in skeletal muscle myocytes. Bone marrow-derived cells developed into β-gal expressing myocytes 2–5 weeks after injection. In contrast, satellite cells, the previously known intramuscular stem cells, were found to differentiate into myocytes after just 5 days. Direct injection of the marrow cells into the muscle was not necessary for the differentiation of marrow cells into myocytes. After whole bone marrow transplantation, skeletal muscle injury was induced, and donor derived marrow cells were found to contribute to the newly formed healing muscle fibers. It is not clear which subpopulation(s) within the marrow contained cells that differentiated into myocytes. The authors tested the adherent and nonadherent subpopulations of the bone marrow separately and both were capable of generating skeletal muscle myocytes.

The potential clinical utility of this finding was demonstrated in a mouse model of muscular dystrophy.13 When affected animals received a bone marrow transplant from wild-type animals, marrow-derived skeletal muscle cells developed, and these genotypically normal cells engrafted and expressed normal dystrophin in up to 10% of muscle fibrils after 12 weeks. While this degree of engraftment is not likely to be adequate to improve muscular function in affected patients, these findings suggest that allogeneic bone marrow transplantation or autologous transplantation of marrow cells that have been modified to correct the genetic defect eventually could be used to treat genetic muscle diseases.

Bone marrow to cardiac muscle

In very promising studies, bone marrow stem cells have been shown to promote repair of myocardial damage following ischemia/reperfusion models of myocardial infarction in mice and rats.1415 The same bone marrow subpopulation that is capable of reconstituting the hematopoietic system after myeloablative bone marrow transplantation was able to differentiate into cardiac myocytes, smooth muscle cells and endothelial cells when injected into the myocardium after ischemic injury.15 A similar study was performed which demonstrated that systemically administered CD34+ human bone marrow cells could also support repair after myocardial infarction in the rat.14 In contrast to the initial studies in the rat, the human bone marrow cells were not injected directly into the infarcted region. Rather, after infarction, they were administered i.v. and traveled to the site of damage where they contributed to healing by contributing to approximately 25% of the newly developed myocardial capillary vasculature. This study demonstrates that the marrow-derived cells do not necessarily need to be injected directly into the site of injury in order to provide a beneficial effect and that the therapeutic effect of these cells could be through induction of endothelial growth, which could promote healing.

A very exciting development in this work has been the demonstration that mobilization of marrow stem cells into the peripheral blood by i.v. administration of a combination of stem cell factor and granulocyte colony-stimulating factor also promoted repair of the infarcted areas.16 Clinical trials in humans are currently under development to test whether growth factor administration following myocardial infarct could prove beneficial to patients. It is important to acknowledge that the mechanisms that may be involved are not yet clear. The improved cardiac functionality with administration of growth factors is probably due to more than just recruitment of cells to become myocytes. There may be positive effects due to increased angiogenesis, decreased apoptosis, as well as other changes. In light of the data obtained using circulating human CD34+ marrow cells, it is likely that the growth factor administration does induce some marrow-derived cells to differentiate into myocardial endothelial cells at the site of injury.

Bone marrow to brain

The major cell types in the brain are the neurons and glial cells, of which there are macroglia (including oligodendrocytes and astroglia) and microglia. Microglia are widely distributed within brain parenchyma and are considered to be resident macrophages within the CNS. (Embryologically, astroglia develop from mesenchymal tissue.) That microglia could derive from bone marrow cells in adults was demonstrated using transplantation of retrovirally marked bone marrow cells in mice.17 In addition to microglia in the brains of these mice, donor-derived GFAP-expressing cells were also detected indicating that marrow-derived cells can also differentiate into astroglia in vivo.17 In 2000, back-to-back papers were published that showed the potential for marrow-derived cells to differentiate into neurons in vivo.1819 One group used GFP expressing donor mice and transplanted the cells i.v. as a bone marrow transplantation.18 The other group used i.p. injection of male wild-type marrow cells into female Pu.1 −/− neonates who lack normal bone marrow function, but had normal CNS development.19 In both studies about 0.2–1% of neurons were calculated to be donor-derived based on co-expression of neuronal markers such as NeuN and either GFP or Y chromosome expression, respectively. It is not yet known which bone marrow subpopulation(s) were capable of this neuronal development. Recent in vitro studies have shown that adherent mesenchymal stem, cells derived from adult bone marrow can differentiate into cells that express neuronal markers.2021

Bone marrow to liver

One of the earliest indications that marrow-derived cells have the capacity to differentiate into epithelial cells of nonmesenchymal origin was a study that showed marrow-derived hepatocytes forming in rats in response to liver damage. Petersen et al22 transplanted male bone marrow into female rats and after donor bone marrow engraftment was confirmed, rats were treated with the chemical toxin carbon tetrachloride and hepatocytic proliferation was inhibited by administration of 2-acetylaminofluorene. Donor-derived Y chromosome positive hepatocytes were found within 2 weeks of liver injury. Soon after this work was published, my colleagues and I used male to female transplantation to demonstrate that marrow-derived cells could differentiate into hepatocytes in vivo in mice23 and in humans24 in the absence of acute hepatic injury. Although it was not clear precisely which marrow-derived cell subpopulation(s) had the potential to differentiate into hepatocytes, we showed that the CD34+lin marrow subpopulation that was enriched for hematopoietic reconstituting ability contained cells that differentiated into hepatocytes and cholangiocytes.23

In one of the most exciting papers to date on the potential of marrow-derived cells to differentiate into functional nonhematopoietic epithelial cells, Lagasse et al25 showed the ability of marrow-derived cells to reconstitute 30–50% of liver hepatocytes and these marrow-derived cells were capable of normalizing the liver function in mutant mice lacking the enzyme FAH which is essential for the metabolism of tyrosine and thus the prevention of tyrosinemia and the build up of hepatotoxic tyrosine metabolites. The FAH−/− recipient animals used cannot survive unless they are treated orally with the medication NTBC that prevents the breakdown of tyrosine to its toxic metabolites. In this study, Lagasse et al performed a bone marrow transplantation using wild-type β-gal-expressing marrow cells and after transplantation, they gradually weaned the animals off the NTBC. When the NTBC was withheld from control FAH−/− mice, all of them succumbed to hepatotoxicity. In contrast, about 30% of the FAH−/− mice that had been transplanted with normal marrow cells survived after NTBC withdrawal and these survivors had 30–50% of their liver mass replaced by β-gal-expressing donor-derived hepatocytes. This study demonstrated clearly that the donor-derived cells that appeared phenotypically to be hepatocytes actually performed all the normal hepatocytic functionality.

Bone marrow to kidney

In order to determine whether bone marrow-derived stem cells can differentiate into epithelial cells of the kidney, mouse and human kidneys were examined after male to female bone marrow transplantation. In both mice and humans, Y chromosome-positive, CD45-negative cells were present in the glomeruli and renal tubules.26 In the human renal grafts, some of the recipient-derived cells within the kidney exhibited a tubular epithelial phenotype, and these cells were identified as epithelial by combining in situ hybridization for the Y chromosome with immunostaining for the epithelial markers CAM 5.2 and the lectin Ulex europaeus. In the mouse kidneys, co-localization of Y-chromosomes and the tubular epithelial markers Ricinus communis and Lens culinaris, and a specific cytochrome P450 enzyme (CYP1A2) identified donor marrow-derived tubular epithelial cells. In a related study, mesangial cells in the glomeruli were found to be marrow derived after wild-type mice were transplanted with syngeneic bone marrow cells from mice transgenic for green fluorescence protein.27 The GFP-positive cells in the mesangium expressed desmin and contracted in response to angiotensin II stimulation. Although mesangial cells have been known to be of mesenchymal origin, it had not previously been shown that these cells could derive from the marrow.

Bone marrow to GI tract, lung and skin

The studies reviewed above have shown that mesodermally derived marrow cells can differentiate into mesodermal (eg muscle), and ectodermal (eg brain) tissue, demonstrating that the boundaries determined by embryologic trilaminar origin are not maintained in the adult. In an effort to determine which bone marrow subpopulation has this plasticity, we performed bone marrow transplants in mice using just a single male-derived marrow cell into each female recipient.28 The marrow subpopulation used was derived by first performing elutriation to select for small round dense cells, then immunomagnetic depletion of lineage-committed hematopoietic cells. The third step was to select those cells which were best able to home to the bone marrow after i.v. transplantation. This was performed by labeling the cells with a fluorescent dye and injecting them into a primary lethally irradiated female recipient After 2 days, the fluorescently labeled donor-derived cells were isolated by FACS from the marrow cells of the female recipient. Single cells from this elutriated, lineage depleted, ‘homed’ population had the potential to differentiate into hematopoietic stem cells and also into mature epithelial cells of the liver, lung, GI tract and skin.28

More questions than answers

The exciting studies reviewed above demonstrate intriguing phenomena and generate many questions. How is a cell's final differentiated state achieved? Is it ever final or can any cell change its gene expression pattern to obtain the phenotype of a different cell? Do the highly plastic marrow-derived stem cells normally play a functional role in tissue repair? Are there disease states that are associated with loss of these highly plastic cells? Does the degree and/or number of highly plastic stem cells change with aging? How is the silencing of some genes and activation of others orchestrated as cells differentiate? Are there shared features of all highly plastic cells? The answers to these questions and many more will require an extensive research effort.

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Correspondence to D S Krause.

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Krause, D. Plasticity of marrow-derived stem cells. Gene Ther 9, 754–758 (2002) doi:10.1038/sj.gt.3301760

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Keywords

  • review
  • hematopoiesis
  • transdifferentiation
  • development
  • bone marrow
  • liver

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