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STEM CELLS IN THE NEWS |
1 Executive Director, Cell Therapy R & D Head, Hematopoiesis Department American Red Cross Holland Laboratory
2 Research Communications Manager American Red Cross Holland Laboratory
Over the past few years, numerous articles have been published in the primary literature in which the authors have reported the ability of various tissue-specific stem cells to develop into cells of unrelated tissue types. This apparent "plasticity" of stem cells from brain, bone marrow, and skeletal muscle has become a holy grail of stem cell therapeutics, promising the eventual clinical use of these adult stem cells in regenerating a cornucopia of damaged tissues [1, 2]. In a previous issue of STEM CELLS, one of us (R.G.H.) espoused the need to put these amazing observations into "Popper perspective" [3]. Now a flurry of papers appear to have done just that, calling into question the concept of adult stem cell plasticity while raising a cautionary note not only with regard to the methodologic limitations of the earlier adult stem cell plasticity studies, but also for the future of regenerative medicine predicated on the postulated use of these cells.
APPARENT TRANSDIFFERENTIATION ATTRIBUTED TO RARE TRANSFORMATION EVENTS OR CELL FUSION
Among the more remarkable stem cell plasticity results in recent years have been those reporting that neural stem cells (NSCs) can transdifferentiate into cells representative of all three germ layers [4–8]. In the February issue of Nature Medicine, Morshead et al. [9] described their efforts to duplicate the studies of Bjornson et al. [4], which were the first to purport that NSCs had hematopoietic differentiative potential. The group transplanted over 128 x 106 cultured neurosphere cells (1 x 106 donor cells per recipient) containing approximately 12 x 106 NSCs into 128 host animals, and analyzed the mice during observation periods of 4-7 months. Following the protocol as described in the original study, donor cells derived from C57BL/6-ROSA26 lacZ transgenic mice (expressing a ß-galactosidase gene) were first injected into irradiated BALB/c mice. Because of the histoincompatibility of this donor-recipient strain combination, the investigators also used congenic C57BL/6 mice as recipients. They examined a total of 31,990 bone marrow colonies from 104 animals injected with 4 independent lines of NSCs, and did not find a single instance of a LacZ+ colony. Morshead et al. next prepared NSCs from C57BL/6J-Gpi1a/a embryos homozygous for the glucose-6-phosphate isomerase-1 (Gpi1) type a isoenzyme marker present in all hematopoietic cells and injected them into 7 C57BL/6J-Gpi1b/b recipients (homozygous for the Gpi1 type b isoenzyme). No Gpi1a/a donor contributions could be detected. Finally, none of 17 C57BL/6J-W41/W41 mice (carrying a mutation in the c-Kit tyrosine kinase that compromises endogenous hematopoiesis) injected with C57BL/6J-Gpi1a/a NSCs were found to have Gpi1a/a donor-type hematopoietic cells, under conditions that should permit detection of 1 cell with hematopoietic-repopulating potential in 1 x 106 NSCs. What the group did observe, however, were marked changes in the growth properties of the NSCs during extended passaging in culture; these included changes in growth-factor dependence, cell-cycle kinetics, cell adhesion, and gene expression. The authors concluded that genetic or epigenetic abnormalities acquired during in vitro culturing of the NSCs may account for the neural-to-blood fate switch originally reported by Bjornson et al.
Two other articles, published in the April 4 issue of Nature, offer further insights into the possible genetic changes that could confer transdifferentiation potential to otherwise lineage-restricted adult stem cells [10, 11]. Both groups set out to examine whether extracellular cues provided by a heterologous microenvironment could alter cell fate decisions of somatic stem cells. Ying et al. co-cultured NSCs with murine embryonic stem cells (ESCs) [10]. The NSCs were derived from two strains of transgenic mice: ZIN40 mice constitutively co-express the lacZ gene and the neomycin (neo) resistance gene; Oct4-GiP mice selectively co-express the green fluorescent protein (GFP) gene and the puromycin (pac) resistance gene in pluripotent and germ line cells under the control of the Oct4 promoter. The ESCs carried the hygromycin resistance-herpes simplex virus thymidine kinase (Hytk) fusion gene inserted into the Oct4 locus by homologous recombination. After 2-4 weeks of culture in selective medium containing G418 or puromycin as appropriate to eliminate the ESCs, cells expressing LacZ (neo) or GFP (pac), respectively, could be recovered from 23 independent co-cultures. Expression of GFP was significant because the Oct4-GiP transgene is not active in NSCs. These results suggested the apparent dedifferentiation of the NSCs to an ESC-like state. However, further investigation revealed that the cells were resistant to hygromycin (indicative of Hytk expression), and analysis of 18 independent isolates documented a tetraploid or near-tetraploid nuclear content. The investigators concluded that "dedifferentiation" of the NSCs occurred through spontaneous generation of hybrid cells and not epigenetic reprogramming of the somatic cell genome due to extrinsic signals.
A companion article by Terada et al. reported the similar ability of murine bone marrow cells to spontaneously fuse with cultured ESCs [11]. Bone marrow cells, obtained from the femurs of female TgN(GFPU)5Nagy transgenic mice co-expressing the GFP and pac genes, were co-cultured with male ESCs. Interleukin-3 (IL-3; also referred to as multipotential colony-stimulating factor) and leukemia inhibitory factor were added to the culture medium to support the growth of primitive hematopoietic precursors and undifferentiated ESCs, respectively. On day 7, IL-3 was withdrawn to suppress the growth of the hematopoietic cells and puromycin was added to eliminate the ESCs. Within 3 weeks, GFP+ puromycin-resistant ESC-like clones could be isolated, which were capable of differentiating into cardiac myocytes in vitro and forming teratomas when injected into nonobese diabetic-severe combined immunodeficient mice. When examined for DNA ploidy, 11/13 ESC-like cell lines demonstrated approximately tetraploid DNA content and the remaining two showed an approximately hexaploid (6n) genotype. Although addition of IL-3 to the cultures was essential for hybrid generation, it is noteworthy that a population of bone marrow cells enriched for hematopoietic precursors (Sca-1+Lin- phenotype; stem cell antigen-1 positive cells lacking lineage differentiation markers), did not increase the frequency of hybrid generation. The authors concluded that hematopoietic stem cells (HSCs) are unlikely to be involved in the cell fusion events they observed, but speculated that their data may provide an alternative explanation for some of the recent transdifferentiation findings.
HEMATOPOIETIC REPOPULATING ACTIVITY IN MUSCLE ATTRIBUTED TO ITINERANT HEMATOPOIETIC, NOT MUSCLE, STEM CELLS
The results of two new articles together with those of an earlier paper also undermine the developing notion of a generalized potential of tissue-specific stem cells. In the February 5 issue of the Proceedings of the National Academy of Sciences, McKinney-Freeman et al. reported that the mononuclear cells from skeletal muscle of C57BL/6-CD45.1 mice, which were capable of repopulating the hematopoietic systems of irradiated C57BL/6-CD45.2 congenic recipients, could be localized exclusively to a population that expresses the CD45 common leukocyte antigen, a specific hematopoietic marker [12]. Further studies in which fractionated CD45+ and CD45- muscle cell populations obtained from C57BL/6-ROSA26 and C57/MlacZ lacZ transgenic mice were evaluated in a muscle injury assay demonstrated that the CD45+ cells contained only limited in vivo myogenic potential. In contrast, CD45- cells incorporated into muscle at high frequency. Taken together, these findings disprove the hypothesis posited by two of the authors previously that putative muscle stem cells with a CD45-Sca-1+c-Kit+ phenotype might account for the hematopoietic repopulating activity in adult skeletal muscle [13]. Using a variety of transplant models—C57BL/6-ROSA26 lacZ mice, C57BL/66TgN (ACTbEGFP)1Osb mice constitutively expressing GFP, the CD45.1/CD45.2 congenic model, and transduction of donor cells with an MSCV-GFP retroviral vector, Issarachai et al. reported on similar findings in the April issue of Experimental Hematology [14]. Like McKinney-Freeman et al., this group only observed hematopoietic reconstitution of irradiated recipients with CD45+ muscle-derived cells. In subsequent experiments, Issarachai et al. obtained additional data that supported earlier work by Kawada and Ogawa demonstrating that the cells in muscle with hematopoietic repopulating activity originate from transplanted bone marrow [15]. In a syngeneic transplant model, the investigators transplanted 9 mice with MSCV-GFP-transduced bone marrow cells. Four to 8 months posttransplantation, the extent of donor reconstitution was determined to be 56%-98%. To stimulate egress of HSCs from the bone marrow and into the peripheral circulation, five of the transplanted mice were subjected to a cytokine-induced mobilization protocol involving administration of G-CSF or G-CSF and Flt3 ligand. Muscle cell suspensions were then prepared after systemic perfusion to avoid blood cell contamination. The mobilization protocol resulted in a modest increase (6.9% versus 3.2%) in the percentage of CD45+GFP+ bone marrow-derived cells recovered from the muscles of the transplanted mice. The data were interpreted to mean that muscle tissue harbors a population of bone marrow-derived cells that arrive there via the circulation, but that the CD45+ cells residing in muscle are not in rapid exchange with those in peripheral blood.
AND THE ANSWER TO THE QUESTION IS
So, is there a future in "plastics?" In aggregate, these new findings reiterate the necessity for scientific rigor in evaluating data from somatic stem cell plasticity experiments. It is important to bear in mind that many of the more celebrated examples of transdifferentiation have been carried out with enriched but not pure populations of tissue-specific stem cells [16, 17]. In these instances, heterogeneity of the population under study may account for the results obtained [18]. In those cases where highly purified adult stem cell populations have been employed [19, 20], reprogramming of the stem cell genomes may certainly have occurred, but the new research suggests that rare transformation events and cell fusion with host cells need to be excluded as possible mechanisms before the issue will be resolved.
Disclaimer:
Any views and opinions expressed herein are those of the authors. They do not necessarily reflect the policies or position of the American Red Cross.
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REFERENCES
This article has been cited by other articles:
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  J. A. Potian, H. Aviv, N. M. Ponzio, J. S. Harrison, and P. Rameshwar Veto-Like Activity of Mesenchymal Stem Cells: Functional Discrimination Between Cellular Responses to Alloantigens and Recall Antigens J. Immunol., October 1, 2003; 171(7): 3426 - 3434. [Abstract] [Full Text] [PDF]
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 Y. Liu, J. Song, W. Liu, Y. Wan, X. Chen, and C. Hu Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res, May 1, 2003; 58(2): 460 - 468. [Abstract] [Full Text] [PDF]
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