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Bone marrow transdifferentiation - fact or artefact?
- The molecular basis of transdifferentiation

Bone marrow transdifferentiation - fact or artefact?

Bone marrow contains two populations of stem cells - (1) haematopoietic stem cells (HSCs), which normally give rise to all mature lineages of blood, and (2) mesenchymal stem cells (MSCs), which can differentiate to bone, cartilage and fat. Previously, bone marrow-derived cells (BMDCs) were thought to be pluripotent and have the ability to transdifferentiate directly to non-haematopoietic cell types [reviewed in 89]. For example, both pancreatic cells [90–92] and liver cells [93–95] have been shown to arise from BMDCs. While these examples were first thought to provide evidence for direct transdifferentiation it is now recognised that (at least in some examples) BMDCs form fusion heterokaryons with cells in the target organs. So far, BMDCs have been shown to fuse with liver, skeletal muscle, cardiac muscle and neurons (Fig. 3) [89]. While there is evidence of metaplasia when BMDCs convert to satellite cells in irradiated or exercise-induced muscle damage [96], there is also evidence that formation of new functional myofibres involves fusion of myeloid cells with the injured muscle fibres [97]. However, one disadvantage of using muscle to study metaplasia is the difficulty in distinguishing between the mature multi-nucleated muscle fibre and nuclei incorporated during fusion.

Criteria for transdifferentiation

In order to unambiguously demonstrate that transdifferentiation has occurred, two criteria must be fulfilled. The first requires that the differentiated state before and after transdifferentiation is demonstrated. The second is to demonstrate the ancestordescendant relationship between the two cell types One approach to addressing the ancestor-descendant relationship is to use the Cre-lox system. In the Cre-lox system, a mouse is engineered to contain two transgenes. Expression of the Cre recombinase is driven by a tissue-specific promoter. A reporter gene is driven by a ubiquitous promoter, but only after the Cre enzyme has excised an inhibitory sequence flanked by loxP sites. This means that the reporter becomes permanently active in the lineages that have previously activated the tissue-specific promoter. The Cre-lox system has been used to detect cell fusion events. Alvarez-Dolado et al. found evidence of BMDC fusion with hepatocytes and cardiomyocytes in vivo [98]. There is also the example of BMDC incorporation into fully differentiated, non-proliferative Purkinje neurons in both humans [99] and mice [98, 100]. If fusion can occur in non-dividing cells, such as Purkinje neurons, or damaged tissues, such as cardiomyocytes following heart failure, there may be potential in the use of cell fusion as a mechanism for cell therapies. While the Cre-lox system is a powerful method for tracing cell lineages, other lineage tracing methods include transplanting male (Y chromosome) donor cells into female recipients or simply labelling the cells of interest with lipophilic dyes such as DiI. Evidence from transplantation studies demonstrates that HSCs can rescue liver function in a mouse model of hereditary tyrosinaemia type I [101]. These mice lack the enzyme fumaryl-acetoacetate hydrolase (FAH) and can normally only survive if treated with the drug 2-(2-nitro-4-trifluoro- methylbenzyol)-1,3-cyclohexanedione (NBTC). HSCs derived from Fah+/+ mice reconstituted the Fah-/- liver by fusing with the existing hepatocytes [102, 103]. The authors excluded the possibility that HSCs from Fah+/+ mice converted to hepatocytes prior to fusion [103] and subsequently established through analysis of the genotype and phenotype of the reconstituted hepatocytes that a specific lineage of HSCs were responsible for fusion. This was confirmed when granulocyte macrophage progenitors (GMP, the colony forming units of progenitors for ganulocytes, macrophages and dendritic cells) or bone marrow-derived macrophages were transplanted into Fah-/- mice, the cells fused with the host hepatocytes and rescued the mutant phenotype [104]. Granulocyte macrophage progenitors may therefore prove potentially useful in the development of therapeutic strategies for cell and organ replacement. Based on these examples, it is important to distinguish between the mechanisms i.e. direct transdifferentiation versus cell fusion. In order to distinguish these possibilities, the number of nuclei or chromosomes in the transdifferentiated cells can be determined. Although tissue damage is thought to be a primary factor for BMDC fusion with target cells, the precise signals regulating fusion and why only some cell types can fuse and others not is still unclear [98]. There is evidence that fused cells become mononucleated again, either by nuclear fusion or by supernumerary nuclei elimination [98, 102]. Whether the fusion product - the stable heterokaryons - can also re-activate post-mitotic, terminally differentiated cell types to undergo cell division is currently under investigation. Despite the limitations of cell fusion rates this mechanism may be considered as a potential avenue for tissue repair.

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