Department of Internal Medicine and Therapeutics, Division of Nephrology, Osaka University School of Medicine, Suita, Japan
Keywords: bone marrow; kidney; side population; SP cell; stem cell
Introduction
The structure of the kidney is the result of a sophisticated interaction between several types of immature cells. Research over the past few decades has elucidated many of the processes underlying cell proliferation, cell fate determination and differentiation. The discovery of genes related to nephrogenesis has been of great help in understanding the sequence of events in nephrogenesis. This information is available at http://golgi.ana.ed.ac.uk/kidhome.html [1]. Attempts to identify further relevant genes are ongoing [24].
It has been a continuing challenge to identify the stem cells involved in the genesis of this complicated organ and to understand how they are regulated. The difficulty begins with the definition of what is a stem cell. Most definitions include as sine qua non requisites the capacity of self-renewal and the potential to generate several different types of differentiated progeny [5]. The criterion of self-renewal implies that when a stem cell divides it can generate one new stem cell and one descendant which further proliferates as a transit-amplifying cell or a rapid-amplifying cell. This property is critical to maintain the pool of stem cells during long periods or even during the life time.
In general, the differentiation potential of the progeny of the most primitive stem cells is increasingly restricted, while specific lineages emerge as the organ develops. Although the kidney is a highly differentiated organ, a population of cells is present that generates new cells either continuously or in response to injury. Confusingly, these cells are often referred to as stem cells because they are thought to constitute a population of cells preserved from the early stage of nephrogenesis. If the repertoire of immature cells in the adult kidney is restricted, so that only renal cells are generated, they should be considered as progenitor cells or committed stem cells rather than true stem cells. In other words, cells that contribute to the repair of the injured kidney are presumably distinct from the stem cells which are involved in nephrogenesis.
Recently, a series of experiments has documented that in different types of adult tissues, some cells can be retrieved that are capable of generating an entire spectrum of cells. For instance, in certain regions of the adult brain, neurons are continuously replaced and added by self-renewing neural stem cells. These cells also generate non-neuronal cells including astrocytes, oligodendrocytes, renal epithelial cells and skeletal muscle cells [69]. Bone marrow-derived cells also give rise to a wide range of non-haematopoietic cells including renal cells in vivo and in vitro [10,11]. Leaving the self-renewal capacity for the moment, these data suggest that immature cells in adult tissues are truly multipotent.
Regulation of stem cells
When a stem cell encounters an environment which provides direct interactions such as epithelialmesenchymal interactions and indirect communication through secreting molecules such as cytokines, an intrinsic differentiation programme of the cell is triggered. To take the example of the kidney, this organ is formed from the metanephrogenic mesenchyme. The ureteric bud arises from the progenitor mesodermal field. As both the metanephrogenic mesenchyme and the ureteric bud communicate with each other in a dynamic reciprocal fashion, multipotent mesenchymal cells differentiate into various types of renal epithelial or non-epithelial cells [12,13]. The cells in the metanephrogenic mesenchyme, however, are special in that the ureteric bud can branch only when the bud enters a cross-talk with the metanephrogenic mesenchyme. In contrast, non-renal tissues, such as the neural tube, enable the metanephrogenic mesenchyme to form renal tubules [14]. It is therefore conceivable that the metanephrogenic mesenchymal cells are totally committed to form renal tissue and are not true stem cells. Intriguingly, neural cells exist along the border of the mesenchyme in the early stages of nephrogenesis [15]. They may be implicated in nephrogenesis sinceas discussed abovethe neural tube induces formation of renal tubules in metanephrogenic mesenchyme.
If we assume that true stem cells exist in the adult kidney, it is necessary that the microenvironment actively inhibits them from taking on unwanted fates. It is assumed that neighbouring cells, so-called niche cells, induce a state of dormancy in stem cells [16]. It remains to be elucidated, however, whether such a control system truly operates in the adult kidney.
Bone marrow as a reservoir
It is possible that fresh stem cells from other tissues are recruited into the kidney. In 1998, Ferrari et al. [17] demonstrated that following injury a proportion of skeletal muscle fibres was actually formed by bone marrow-derived cells. This was documented in chimeric animals following transplantation of genetically marked bone marrow cells. Currently, it is widely accepted that bone marrow-derived cells can give rise to a wide range of differentiated cells such as hepatocytes, cardiomyocytes and smooth muscle cells. For the adult kidney, Imasawa et al. [18] and Ito et al. [19] independently reported that bone marrow-derived cells have the potential to differentiate into glomerular mesangial cells in mice or rats. Repopulating mesangial cells seem to enter the glomerulus through the juxtaglomerular zone [19,20]. Bone marrow-derived cells also produce renal epithelial cells in vivo and this has also been found in humans [2123]. The fact that a single bone marrow-derived cell (or a fraction of purified haematopoietic cells) can give rise to multiple types of epithelial cells, strongly suggests that multipotent stem cells are present in the adult bone marrow [21,24].
The bone marrow is a heterogeneous tissue comprising haematopoietic cells, macrophages, sinusoidal endothelial cells, stromal cells and fat cells. Furthermore, there is a relevant type of stem cell in bone marrow: mesenchymal stem cells, presumably related to stromal fibroblastic cells, that provide biological and physical support to haematopoietic stem cells in the marrow. In the 1970s, Friedenstein et al. [25] established a culture of fibroblastic adherent cells from bone marrow. Later, Prockop [26] and Pittenger et al. [27] found that the fibroblastic adherent cells include mesenchymal stem cells which are capable of producing mesenchymal tissues such as bone, cartilage, fat and smooth muscle. Such fibroblastic cells are often identified with the marrow stromal cells. When bone marrow is cultured using a different protocol, conditions can be found which support haematopoiesis in vitro with high efficiency [28]. Iwatani et al. [29] successfully converted rat bone marrow-derived adherent cells into mesangial-like cells in vitro by using platelet-derived growth factor-BB and collagen type IV. In the context of the previous report on glomerular remodelling [19], this would suggest that mesenchymal stem cells in the adult bone marrow are capable of producing mesangial cells in vivo, at least under pathological conditions. It should be noted, however, that a controversy continues as to whether marrow stromal cells are transplantable or whether marrow stromal cells circulate in blood just like mobilized haematopoietic stem cells [30]. It is possible that there is no strict borderline between haematopoietic stem cells and mesenchymal stem cells, but that gradual transitions exist. Recently, Reyes and Verfaille [31] and Jiang et al. [32,33] established multipotent adherent stem cells from human and rodent bone marrow stromal cells. These cells have been named multipotent adult progenitor cells (MAPCs). They proliferate without showing signs of senescence in vitro. They are able to differentiate not only into blood cells, but also into mesodermal, neuroectodermal and endodermal cells.
Collectively, the adult bone marrow seems to harbour stem cells which contribute to the generation of non-haematopoietic somite cells. Nevertheless, it remains unknown whether the bone marrow-derived stem cells have to be recruited de novo into the adult kidney or whether they are present in the kidney in a dormant state.
Side population (SP) cells
An intriguing method to purify haematopoietic stem cells from the murine bone marrow was reported in 1996 [34]. This technique does not rely on surface markers that are generally used to isolate haematopoietic stem cells, but on the differential efflux of a vital DNA binding dye, Hoechst 33342. Because of its unique fluorescence pattern during flow cytometry analysis, the cells are referred to as side population (SP) cells or as Hoechstlow cells. The SP cells, which are mostly CD34-negative, are small and round in shape and can be obtained from non-haematopoietic tissues [3537]. One of the multidrug-resistance genes, ATP-binding cassette superfamily G (white) member 2 (ABCG2), is mainly responsible for the dye efflux [38]. Because SP cells can be obtained from almost all species and adult tissues tested so far, and because they are multipotent, it is assumed that the efflux of Hoechst 33342 dye is a common trait of multipotent tissue stem cells [36,37,39,40].
Iwatani et al. [41] purified SP cells from adult rat kidney and tested their differentiation potentials. When kidney-derived SP cells were administered by intravenous injection into irradiated rats, the engrafted cells contributed to hepatocytes, skeletal muscle cells and haematopoietic lineages, but not to renal cells including mesangial cells and renal epithelial cells. This was true even after inducing renal injury in the Thy1 glomerulonephritis model, where, following injury, no transplanted SP cells lodged in the kidney. These results imply that the SP cells in the adult rat kidney are not engaged via renal tissue-specific interactions. Obviously they are not committed to the kidney. Additionally, it is unlikely that bone marrow-derived mesangial cells are the progeny of SP cells in the kidney. According to their data, at least some SP cells in the kidney are derived from bone marrow.
SP cells represent a heterogeneous cell population. Skeletal muscle contains two types of SP cells: CD45+ and CD45-. Expression of CD45 is an index of haematopoietic lineage. At present, it is known that CD45+ SP cells contribute to haematopoietic lineages, while CD45- SP cells can differentiate into non-haematopoietic lineages including skeletal muscle [37,40]. Regardless of the expression of CD45, it remains unknown whether SP cells in non-haematopoietic tissues are totally derived from bone marrow, and whether they are constitutively replaced.
Perspective
In 2002, Terada et al. [42] and Ying et al. [43] reported that immature cells are capable of fusion with other types of cells in vitro. After fusion, the immature cells adopt the gene expression corresponding to a tetraploid (4n) cell [42,43]. If such fusion events occur in vivo, the interpretation of experiments using transplantation of immature stem cells will become problematic. For example, expression of a transgene in the skeletal muscle might simply reflect cell fusion, and could not be taken as evidence of differentiation from a stem cell. In the case of skeletal muscle cells, fusion of nuclei is not necessary to incorporate the transgene because the skeletal muscle fibre is a multinucleated cell.
Despite recent progress in stem cell biology, an important question remains unresolved, namely whether each of the different cell lineages in the adult kidney has its own progenitor cells or whether there are universal stem cells residing within the kidney or elsewhere capable of generating different renal cell types. It is still unknown whether immature stem cells have been set aside or not during nephrogenesis for postnatal repair and remodelling of the kidney. Nevertheless, for the clinician the exciting perspective is provided that bone marrow-derived cells can be put to therapeutic use to repair various types of injured tissues, even though it remains unclear at present whether they are physiological stem cells or not.
Conflict of interest statement. None declared.
Notes
Correspondence and offprint requests to: Takahito Ito, MD, Department of Internal Medicine and Therapeutics, Division of Nephrology, Osaka University School of Medicine, Box A8, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Email: taka{at}medone.med.osaka-u.ac.jp
References
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