The term side population (SP) was coined to refer to the vital dye-effluxing population of cells within the murine bone marrow which were found to be greatly enriched for long-term repopulating stem cell activity. The use of fluorescence sorting for dye efflux activity became a method for the enrichment of hematopoietic stem cells, with their ability to efflux vital dyes stemming from the expression of transmembrane proteins of the ABC transporter superfamily. The subsequent observation of ABC transporter-positive dye-effluxing populations with stem cell properties within a wide variety of solid organs has fueled the promise of SP-based regenerative medicine for tissues including heart, cartilage, vasculature, pituitary and kidney. A non-hematopoietic endogenous SP has been identified in the kidneys of bony fish, rodents and humans. This renal SP demonstrates multipotency, a capacity to integrate into renal tubules in response to injury and a modest capacity to improve renal function after acute experimental damage. This chapter describes the multipotency, regenerative capacity, origin and mechanism of action of the kidney SP, and discusses the potential of such utilizing populations in regenerative nephrology.

What is the Side Population?

The isolation of a stem or progenitor population can rely on a combination of cell surface markers, enrichment based on location and/or isolation based on functional properties. While the mouse hematopoietic stem cell (HSC) population is often defined on the basis of lack of expression of a series of lineage epitopes, and positive expression of canonical HSC markers such as Sca-1 and c-Kit (CD117), one phenotypically defining property of HSCs is low fluorescence after staining with vital dyes, including Rhodamine 123 and Hoechst 33342.

The term Hoechst “low” side population (SP) was originally described in murine bone marrow preparations where isolation based on dye efflux resulted in 1000–3000-fold enrichment for long-term repopulating HSC activity in bone marrow transplantation assays . This proved as effective as the isolation of HSCs based on sorting for the commonly used combination of lineage cell surface markers . As a result, the application of this dynamic measurement alone or in combination with antibody staining has proven to be a powerful tool in the purification of HSCs from mouse bone marrow.

How and why do these cells efflux dyes? Supravital dyes, including Hoechst 33342, bind to AT-rich regions within the minor groove of DNA in all cells . Hence, the resulting cellular fluorescence intensity is an index of DNA content, and can discriminate between different stages of cell cycle . The efflux of such dyes from a cell results from the activity of membrane efflux pumps, all of which are members of the ATP-binding cassette (ABC) transporter superfamily. ABC transporters are ATP dependent and can move a variety of cargoes into or out of cells, including metal ions, amino acids, sugars, peptides, hydrophobic compounds, inorganic anions, metabolites, xenobiotics and other drugs . Some ABC transporters are present on the plasma membrane, while others exist in subcellular membrane compartments such as Golgi and endoplasmic reticulum (ER). ATP transporters are widely expressed throughout the mammalian body, although predominantly in excretory organs including the liver, placenta, kidney, intestine and blood–brain barrier . The ABC transporters most commonly associated with the dye-efflux properties of the SP include multidrug resistance 1/P-glycoprotein (Mdr1a/1b – mouse/MDR1 – human) and breast cancer resistance protein 1 (Bcrp1)/ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2) . While dye efflux across the plasma membrane may result from other non-ABC transporters within the plasma membrane, ABC transporter-mediated efflux is specifically blocked by calcium channel blockers such as verapamil or reserpine and inclusion of these compounds in the staining media ablates the SP phenotype.

Role of ABC Transporters: Markers or Mediators of Stem Cells

Given the widespread expression and functional roles of ABC transporters, it may seem unlikely that their presence alone confers a stem cell phenotype. However, enforced expression of at least some of these membrane transporters using retroviral vectors has been shown to have direct functional effects on murine HSCs. Induced expression of ABCG2 in HSCs blocked hematopoietic development , whereas overexpression of MDR1 resulted in HSC expansion and myeloproliferative disorder . This suggests that the expression of these transporters may assist in determining cellular differentiation. While the presence of such transporters may contribute to the SP phenotype, they are not exclusive markers that would allow purification based on their presence. In human bone marrow, ABCG2 coexpression with the HSC markers CD34 and CD133 was observed at low or undetectable levels and cells sorted solely on the marker ABCG2 had little colony-forming potential in vitro . This may be due to the presence of multiple ABC transporters on the stem cell population.

A recent study of the SP in the murine myocardium across development suggests that the ABC transporter primarily responsible for this phenotype shifts from Abcg2 during development to Mdr1 in postnatal life . This suggests a distinction between embryonic and adult cardiac SP but does not eliminate the possibility that the two are in the same lineage. The functional significance of Abcg2 in embryonic cardiac SP, however, appears to be in maintaining proliferative capacity and cell survival as loss of this gene results in cell death and reduced division . Similarly, in embryonic stem cells, Abcg2 plays a role in the maintenance of pluripotency . In line with this is the observation that during early murine development, all cells of the morula and blastocyst inner cell mass are positive for Abcg2 and Abcb1/P-glycoprotein, whereas this expression is lost as cellular pluripotency gives way to lineage commitment .

It has been proposed that efflux of drugs and toxins via these pumps provides protection to the stem cell niche. Indeed, hypoxia can apparently increase the expression of Abcg2 in the cardiac SP. Martin et al. showed this allowed the SP cells to consume hydrogen peroxide after ischemic injury. Increased Abcg2 was elicited in response to hypoxia-inducible factor-2α (HIF-2α) and was accompanied by increased α-glutathione reductase expression, thereby endowing a cell survival benefit in the face of elevated hydrogen peroxide . What appears to occur within the heart is an increase in SP as a percentage of total heart tissue. However, hypoxia may not be causing an increase in SP fraction but simply increasing the levels of expression of Abcg2 on all cells.

Existence of Side Population from Other Organs and their Association with Organ Regeneration

SP cells have now been isolated from a wide variety of mammalian tissues including skeletal muscle, lung, liver, heart, testis, kidney, skin, brain, fat, uterus and mammary gland ( Table 11.1 ). In some instances, the existence of these populations has been verified in humans. The diversity of tissues harboring SP has largely been reviewed previously , although further populations have now been identified, as has evidence of a link with tissue repair and regeneration and/or the presence of stem cell characteristics. The tracheobronchial epithelium of the human lung contains an SP fraction which increases in proportion dramatically in asthmatic lungs . These cells showed stable telomere length and were able to be cultured for 16 passages. These cells were also able to form a differentiated epithelium at an air–media interface, suggesting regenerative capacity within the lung. An SP can be isolated from the dental pulp and subfractionation of these cells to obtain the CD31 ⁻ /CD146 ⁻ cells yielded a population of cells able to elicit a total regeneration of the pulp and accompanying vasculature in a canine model of pulp amputation . These cells appear to produce proangiogenic factors, suggesting that a tropic activity was responsible for recruitment of endothelial cells rather than transdifferentiation .

To test the ability of skeletal muscle-derived SP to enhance regeneration, GFP-positive myoblasts were transplanted into immunocompromised normal mice or dystrophin-deficient mice with or without accompanying CD31 ⁻ CD45 ⁻ SP. The presence of the SP fraction increased the number and distribution of GFP-positive myoblasts integrating into the recipient muscle as well as stimulating their proliferation . The SP was shown to produce matrix metalloproteinase-2 (MMP-2), gelatinase and a number of proliferative cytokines, and it was subsequently shown that MMP-2 alone was responsible for enhanced myoblast migration in vivo . MMP-2 activity has also been shown to be important in stem/progenitor cell differentiation. In the tooth this results from the MMP-2-mediated cleavage of dentin matrix protein (DMP1) which then acts directly on the dental pulp progenitors . Another group has shown that the SP itself produces the DMP1, hence these cells induce the production of MMP-2 by adjacent cells and provide the substrate to be activated . Others propose that in the heart the MSCs increase the generation of MMP-2 by the surrounding cardiac fibroblasts .

An SP also exists within the stromal compartment of human adult adipose tissue . These cells showed the anticipated mesodermal multipotentiality in vitro but also facilitated scar-free wound healing in vivo . SPs also exist in articular cartilage , vocal cord and pituitary gland .

Does Dye Efflux Equal Stem Cell?

The presence of SP within a variety of solid organs has raised the possibility that low fluorescence following Hoechst staining may represent a common stem cell characteristic. However, other than dye efflux, these populations vary significantly. SP from solid organs all display high expression of Sca-1 and the absence of hematopoietic lineage markers . However, other than these few examples, there is little commonality of epitopes between SP from different organs. Indeed, it would appear that each organ contains a distinct SP in terms of cell surface marker profile.

To be defined as stem cells, SP cells must possess the capacity to differentiate into multiple cell lineages contained in their tissue of residence. For example, murine bone marrow SP represent long-term repopulating HSCs able to form all hematopoietic lineages. Arguably only the bone marrow SP fraction has been stringently assessed for this capacity. This may result from the current limitations in suitable assays for tissue-derived stem cells rather than the potential of the SP they contain. As well as being able to generate cell lineages within their tissue of residence, bone marrow SP have the capacity to differentiate into skeletal muscle , liver , osteogenic and cardiac cells in vivo. Skeletal muscle SP has been reported to give rise to blood derivatives and skin SP to skeletal muscle cells . The hematopoietic capacity of liver SP cells in vitro was less robust .

Liver SP cells are able to give rise to a variety of liver-specific cell types, including mature hepatocytes and bile duct epithelium . Welm et al. tested the differentiative potential of mammary gland SP cells after isolation from Rosa26 transgenic mice and transplantation into cleared fat pads of immunocompromised Rag-1 ^−/− recipient mice . Donor SP cells were found contributing to both the ductal and alveolar epithelium of the resulting mammary outgrowths . Finally, SP derived from the testis was able to generate the full range of spermatogenic stages when delivered into testes of busulfan-treated mice .

In a study of the adipose SP, gene expression at point of isolation identified the expression of a number of stem cell markers (Notch pathway and early vascular precursor genes) as well as the expression of α-smooth muscle actin (α-SMA), CD34, Angpt2, Flk1, CD31, VE-cadherin and c-met . The authors suggest that this points to a pericytic origin for the adipose SP and show that such cells can undergo myogenic and vasculogenic differentiation in vitro and in vivo, as has been reported for other vessel-associated cells. This is reminiscent of the mesenchymal stem cell (MSC) populations of many organs . In most instances, the relationship between MSCs and SP from a variety of organs has not been well investigated. However, a common pericytic location may suggest an overlap in these populations in some instances. In the lung, it has been shown that the CD45 ⁻ SP contains MSCs in as far as these cells have a capacity to clonically proliferate with stable telomere length, show expression of CD44, CD90, CD105, CD106, CD73 and Sca1, lack classical hematopoietic markers and differentiate into mesenchymal lineages in vitro . Hence, either the line between these various cell definitions is quite blurred or the assays used to distinguish them apart in vitro do not make this distinction.

Other SP have been described as being able to differentiate into endothelium. Adult myocardial SP treated for 4 weeks with vascular endothelial growth factor-A (VEGF-A) in vitro formed vWF-positive endothelial cells . A vasculogenic claim has been made for a subfraction of the dental pulp SP , where parallels were drawn between endothelial progenitor cells (EPCs) and the CD31 ⁻ CD146 ⁻ fraction of the SP. These cells were also CD34 ⁺ , Flk1 ⁺ and CD45 ⁻ and produced VEGF-A, GCSF, GM-CSF and MMP3; however, no evidence was presented to show that these cells themselves became the newly formed vasculature in vivo.

Fusion Versus Transdifferentiation: Does it Matter?

Despite the data presented here suggesting multilineage potential for a variety of SP populations, whether this represents transdifferentiation or fusion with another differentiated cell type needs to be assessed carefully. Indeed, what is most critical is whether or not these proposed SP-derived cell types are functional. This has been investigated using the genetic dystrophin-deficient mdx mouse strain. Skin SP cells from male donor mice when injected into female mdx mice generated dystrophin-expressing Y-chromosome-positive muscle fibers 3 months after transplant . Similarly, in autologous transplantation of muscle SP cells lentivirally transduced with the human dystrophin gene, cells were detected exclusively in the muscles of mdx5cv mice after reintroduction via the vasculature , again suggesting active homing of these cells to sites of damage . Such studies have substantial implications for autologous cell therapy for genetic muscular disorders and potentially also for other human single gene diseases including cystic fibrosis and Huntington’s disease.

Another way to assess claims of SP transdifferentiation potential is to compare this fraction to non-SP fractions within the same organ. Synovial-derived SP cells can respond to BMP7 to produce collagen type II, suggesting the potential to become cartilage. However, other non-SP cells within the synovium also responded to BMP7 in this way , arguing against SP enriching for a stem cell fraction in this organ. Indeed, the ATDC5 chondroprogenitor line contains both SP and non-SP fractions. The SP fraction increases as a percentage of the total in response to hypoxia; however, this fraction does not show any greater chondrocytic potential than the non-SP fraction .

While evidence for SP transdifferentiation after in vivo infusion is variable, infusion of SP does result in these cells reaching injured organs, suggesting that homing of SP can occur. Alvarez-Dolado et al. showed that the ensuing engraftment of whole bone marrow from genetically marked reporter mice transplanted into lethally irradiated recipients occurred through fusion of host and donor cells and not through transdifferentiation. Here, multinucleated cells containing donor nuclei were observed in the Purkinje cells of the brain, hepatocytes and cardiac muscle . Subsequently, it was demonstrated that myelomonocytic progeny of a single SP cell engrafted via a fusion event as opposed to direct differentiation into tissue-specific progenitors or mature organ-specific populations . In the case of studies in which donor cells integrated into mdx muscle, the mechanism of apparent integration is harder to establish as myofibers are multinucleated. Revertant muscle function would appear to suggest that, irrespective of whether fusion or transdifferentiation had occurred, functional dystrophin was present. However, even this is complicated by the fact that dystrophin expression in a proportion of myofibers can occur in such mice without transplantation of wild-type cells as a result of exon skipping . This becomes important when considering data using irradiated Scgd ^−/− mice (mutation that causes loss of δ-sarcoglycan expression, producing progressive cardiomyopathy and muscular dystrophy) transplanted with bone marrow SP cells, where engraftment with donor cells was observed but no expression of δ-sarcoglycan resulted .

Origin of Non-Bone Marrow Side Population Cells

There is accumulating evidence to suggest that SP within the bone marrow or circulation can be recruited to sites of tissue damage, including skeletal muscle , heart , liver and kidney , during regeneration from certain types of damage. This raises the question of whether SP fractions in specific organs originate from a common pool of cells in the bone marrow and adopt tissue-specific characteristics upon seeding within a specific local environment. There are conflicting data relating to this possibility. In lethally irradiated recipients transplanted with bone marrow SP cells isolated from Rosa26 transgenic mice, 5 months after transplant approximately 40% of host muscle SP cells were LacZ positive, thus being derived from donor cells . Total bone marrow and bone marrow SP cells transplanted into lethally irradiated mice produced lung SP cells that were donor derived and contained both CD45 ⁺ and CD45 ⁻ fractions . In lethally irradiated mice transplanted with bone marrow SP cells, donor cells contributed to formation of both CD45 ⁺ and CD45 ⁻ hepatic SP populations after DDC treatment . While these confirm an ability for bone marrow SP to home to sites of injury, they do not prove that all organ SP cells are derived from the bone marrow.

The alternative possibility is that organ-specific SP cells arise solely as a consequence of the normal development of that specific organ, but share the SP phenotype as a function of their inherent biological characteristics. This has been established for the testis, where 8 months after transplantation of GFP-positive bone marrow into germ cell-deficient mice ( W54/WV ), the recipient testis SP remained a similar size and contained only negligible GFP-positive cells . In contrast, 50–60% of the CD45 ⁺ cells in spleen and > 70% of the bone marrow SP were GFP positive, indicating successful engraftment into the hematopoietic compartment, as would be expected . Of note, an analysis of the SP fraction within the liver across embryonic development suggests that this population is likely to have an initial origin in common with the oval cell progenitors of the liver, a population that has long been established as playing a role in liver regeneration .

Heterogeneity Within Distinct Side Population Fractions

While any potential stem cell population can be greatly enriched via specific selection protocols, the resultant cell populations remain heterogeneous. This is also the case for SP. While lack of CD45 is regarded as a feature of tissue SP fractions, approximately 75% of murine liver SP cells express CD45. The liver SP cells with the highest efflux capacity are enriched for CD45 ⁻ cells , but both CD45 ⁺ and CD45 ⁻ subpopulations express the stem cell markers CD34, c-kit, Sca-1 and Thy-1 . Similarly, while the lung SP represents 0.05–0.07% of the total viable cell population within the lung, 60–70% of these cells are CD45 ⁺ with the remainder being CD45 ⁻ . While both lung SP fractions were Sca-1 ⁺ Lin ⁻ , the CD45 ⁺ fraction was CD34 ⁺ CD31 ⁺ while the CD45 ⁻ fraction was CD34 ⁻ CD31 ^+/− .

Within the literature, there are also large discrepancies between reported SP abundance within the same tissue. These are likely to reflect variations in isolation stringency and hence overall heterogeneity. In muscle, the fastest effluxing SP cells are almost exclusively Sca ⁻ 1 ⁺ CD45 ⁻ . However, if the stringency of isolation is dropped by increasing the gate size during sorting to include SP cells closer to the main population (MP), the proportion of Sca-1 ⁻ CD45 ⁻ and Sca-1 ⁻ CD45 ⁺ cells significantly increases . This observation has also been made with respect to the bone marrow, where the SP cells shifted from a Sca-1 ⁺ CD45 ⁺ phenotype to a Sca-1 ⁻ CD45 ⁺ as the size of the SP gate increased . It should be noted that the original protocols for isolation of SP cells from bone marrow demonstrated a population that was phenotypically (Lin ⁻ CD45 ⁺ Sca-1 ⁺ ) and functionally quite homogeneous in terms of long-term reconstituting HSCs owing to a high level of stringency in the application of the sorting.

Clearly, variation in the level of SP heterogeneity, due to either inherent heterogeneity and/or variations in stringency of isolation between studies, makes the functional assessment of relative enrichment critical. This highlights the need to distinguish between “side population cell” and “stem cell” as the latter may represent only a fraction of the former. An assessment of the stem cell capacity of such cells in vitro and/or in vivo must be employed both to optimize the conditions for isolation of SP cells from individual tissue and to assess the relative enrichment afforded by sorting based on dye efflux. Ultimately, the subfraction of any SP responsible for any observed functional activity may not be able to be definitively identified.

Identification of Side Population Cells Within the Embryonic and Adult Kidney

As for many other organs, the presence of an SP has also been reported in the postnatal kidneys of mouse , rat and human . An SP fraction was also isolated from embryonic kidney . The kidney as an organ is rich in ABC transporters, given the role of this organ in water and ion flux. Distinct segments of the nephron express distinct ABC transporters depending on their role in excretion and water reclamation. It is not surprising, therefore, that a dye-effluxing population exists within the kidney or that at a proportion of this renal SP is epithelial in nature. However, the accompanying observations regarding the in vitro and in vivo properties of the renal SP and the effect on renal function on the infusion of this population suggest that the presence of a renal SP does not simply reflect an abundance of ABC transporters in the kidney.

While the existence of such a population in kidney was agreed, there were large discrepancies with respect to the abundance of this fraction between different studies. These discrepancies are likely to have resulted from slight variations in tissue dissociation, time, temperature and dye concentration from one experiment or laboratory to the next . For example, the few studies that have examined the murine kidney SP report dramatically different percentages of this population. Asakura and Rudnicki and Hishikawa et al. determined that the SP fraction represented approximately 5% of all total viable cells from the adult mouse kidney. In contrast, Challen et al. report the frequency of the same population to be 0.1%, which is similar to the incidence of SP from adult rat kidney reported by Iwatani et al. of 0.03–0.1% and more in line with the SP frequency more typically associated with other organs. This again highlights the variability in this staining procedure.

SP within the kidney is not confined to mammals. The existence of an SP has been reported within teleosts, including the zebrafish and Gibuna cruian (carp) . Hematopoiesis in these organisms is maintained in the kidney, hence this may reflect the HSC fraction of these organisms and not represent an equivalent to the SP of the mammalian kidney. Indeed, the properties of teleost kidney SP were equivalent to those of HSCs with respect to long-term repopulation capacity .

Gene Expression Profile and Immunophenotype of Kidney Side Population

Several studies have investigated the gene expression profile and immunophenotype of the kidney SP to understand their function and assist in the subfractionation of these heterogeneous populations based on the identification of unique genes encoding cell surface proteins. Using gene expression profiling, Hishikawa et al. identified musculin/MyoR as a novel marker of adult murine kidney SP cells; however, this gene was not expressed in the study of Challen et al. . Challen et al. observed significant differences in gene expression between kidney and bone marrow populations, but a high congruence of expression between adult and embryonic kidney SP ( Fig. 11.1A ). The genes upregulated in both embryonic and adult kidney SP samples compared to bone marrow included a number of key growth factor receptors, including epidermal growth factor receptor (EGFR) and growth hormone receptor (GHR). The latter is of note given the potential use of exogenous GH therapy in the treatment of end-stage renal disease patients . In addition, EGF is known to be a potent regulator of stem cell activity. Kidney SP cells also expressed CD24 and CD133. CD133 has been described as a marker of human hematopoietic and neural stem cells and reportedly marks a human adult kidney stem cell population that may contribute to the repair of renal injury . Genes that were upregulated in bone marrow SP cells compared to both kidney SP samples included known hematopoietic and HSC markers (CD34, CD44, CD45, CD53, c-kit, Ly64).

Affymetrix microarray data analysis. (A) Heat map of Affymetrix gene expression in bone marrow, adult and embryonic kidney shows high congruence between kidney samples. Right-hand panel shows relative expression of all genes in kidney side population (SP) compared to bone marrow SP with red representing strongly upregulated and green representing strongly downregulated kidney SP genes. (B) Identification of “common SP genes” or a putative SP molecular signature by determining the genes with less than 25% variance in signal between bone marrow, E15.5 and adult kidney SP samples. The Venn diagram demonstrates a common signature of 475 genes. The right-hand panel illustrates the relative expression of these 475 genes across all three samples. Please see color plate at the end of the book.

Using immunophenotyping, kidney SP shared some characteristics with bone marrow SP cells, such as high expression of Sca-1 and CD24. However, the kidney SP lacked the canonical HSC marker c-kit ( Table 11.2 ). This again supported the premise that the renal SP is an endogenous population and not a circulating hematopoietic contaminant. In contrast, the only obvious difference in phenotype between the adult and embryonic kidney SP was an increased CD31 antigen in the adult kidney SP fraction.

Table 11.2

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