Masaki Nishikawa

Stepwise renal lineage differentiation of mouse embryonic stem cells tracing in vivo development

The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24(+) cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III.

Lithium induces c-Ret expression in mouse inner medullary collecting duct cells

We found in our present study that lithium (Li(+)) induced the expression of endogenous c-Ret, a tyrosine kinase receptor, in murine inner medullary collecting duct (mIMCD-3) cells. Delineation of the promoter region required for the effect of Li(+) identified a positive regulatory element within 180bp upstream of the transcription initiation site. This region contained three putative GC-rich Sp1 binding sites found to be essential for c-Ret induction by Li(+). The effect of Li(+) was mediated through glycogen synthase kinase 3β (GSK-3β) inhibition, although there was no biding site for T cell factor/lymphoid enhancer factor (TCF/LEF) in the 180bp. We found that Li(+) activated the mammalian target of rapamycin (mTOR) pathway via GSK-3β in these cells, and the effect of Li(+) to induce c-Ret was amenable to the inhibitory effect of the mTOR inhibitor, rapamycin. We also found that alterations in both cellular β-catenin levels and mTOR activities affected the effect of Li(+) on c-Ret transcription in a cooperative manner. In summary, our results show that Li(+) can induce c-Ret expression in mIMCD-3 cells through both β-catenin- and mTOR-dependent pathways downstream of GSK-3β inhibition, which act synergistically on the GC-rich Sp1 binding elements in the promoter region.

Engineering of Implantable Liver Tissues

In this chapter, from the engineering point of view, we introduce the results from our group and related research on three typical configurations of engineered liver tissues; cell sheet-based tissues, sheet-like macroporous scaffold-based tissues, and tissues based on special scaffolds that comprise a flow channel network. The former two do not necessitate in vitro prevascularization and are thus promising in actual human clinical trials for liver diseases that can be recovered by relatively smaller tissue mass. The third approach can implant a much larger mass but is still not yet feasible. In all cases, oxygen supply is the key engineering factor. For the first configuration, direct oxygen supply using an oxygen-permeable polydimethylsiloxane membrane enables various liver cells to exhibit distinct behaviors, complete double layers of mature hepatocytes and fibroblasts, spontaneous thick tissue formation of hepatocarcinoma cells and fetal hepatocytes. Actual oxygen concentration at the cell level can be strictly controlled in this culture system. Using this property, we found that initially low then subsequently high oxygen concentrations were favorable to growth and maturation of fetal cells. For the second configuration, combination of poly-L: -lactic acid 3D scaffolds and appropriate growth factor cocktails provides a suitable microenvironment for the maturation of cells in vitro but the cell growth is limited to a certain distance from the inner surfaces of the macropores. However, implantation to the mesentery leaves of animals allows the cells again to proliferate and pack the remaining spaces of the macroporous structure, suggesting the high feasibility of 3D culture of hepatocyte progenitors for liver tissue-based therapies. For the third configuration, we proposed a design criterion concerning the dimensions of flow channels based on oxygen diffusion and consumption around the channel. Due to the current limitation in the resolution of 3D microfabrication processes, final cell densities were less than one-tenth of those of in vivo liver tissues; cells preferentially grew along the surfaces of the channels and this fact suggested the necessity of improved 3D fabrication technologies with higher resolution. In any case, suitable oxygen supply, meeting the cellular demand at physiological concentrations, was the most important factor that should be considered in engineering liver tissues. This enables cells to utilize aerobic respiration that produces almost 20 times more ATP from the same glucose consumption than anaerobic respiration (glycolysis). This also allows the cells to exhibit their maximum reorganization capability that cannot be observed in conventional anaerobic conditions.

Simultaneous evaluation of toxicities using a mammalian cell array chip prepared by photocatalytic lithography

A prototype of a mammalian cell array chip was developed on a flat glass surface. A superhydrophilic (water contact angle=5 degrees)/highly hydrophobic (120 degrees) pattern was prepared on a fluorinated polymer-coated glass surface by means of photocatalytic lithography, and A549 (a human alveolar epithelial cell line), Hep G2 (a human hepatoma cell line) and mouse fibroblast 3T3 cells were inoculated onto the superhydrophilic regions. The cell populations were confined in the superhydrophilic regions for at least 24 h and separated from each other for at least one week. Organ-specific toxicity of aflatoxin B(1) and non-specific toxicity of adriamycin were successfully detected by using the cell array chip.

In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells

Summary A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys.

Controlled tubulogenesis from dispersed ureteric bud‐derived cells using a micropatterned gel

Developmental engineering is a potential option for neo‐organogenesis of complex organs such as the kidney. The application of this principle requires the ability to construct a tubular structure from dispersed renal progenitor cells with defined size and geometry. In this present study we report the generation of tubular structures from dispersed ureteric bud cells in vitro by using a micropatterned gel. Dispersed CMUB‐1 cells, a mouse ureteric bud‐derived cell line, or mIMCD cells, a mouse collecting duct‐derived cell line, were suspended in collagen I and seeded into an agarose‐based micropatterned gel. We found that within 24–36 h of incubation, the cells developed a tubular structure that conformed to the geometry of the micropattern of the gel. The lumen formation of the tubular structure was confirmed by immunohistochemical staining and observed by confocal microscopy. We found that higher concentrations of collagen I negatively influenced the efficiency of tubular formation. Tubule formation in CMUB‐1, but not mIMCD, cells was positively influenced by the addition of aldosterone (10, 50 and 200 µg/ml), FGF (50 and 100 µg/ml) and fibronectin (10 and 50 µg/ml) to the growth medium. We further demonstrated the functionality of the generated tubes by in vitro budding, which was induced by growth factors, such as glial cell‐derived neurotrophic factor (GDNF) or fibroblast growth factor 7 (FGF7), in the presence of beads soaked with the activin A inhibitor follistatin. Our current study thus demonstrates the possibility of constructing a functional tubular structure from dispersed ureteric bud cells in vitro in a controlled manner. Copyright

Effective induction of cells expressing GABAergic neuronal markers from mouse embryonic stem cell.

Successful derivations of specific neuronal and glial cells from embryonic stem cells have enormous potential for cell therapies and regenerative medicine. However, the low efficiency, the complexity of induction method, and the need for purification represent obstacles that make their application impractical. In this study, we found that PDGFRα+ cells derived from mouse embryonic stem cells (mESC) can serve as a useful source from which to induce cells that express γ-aminobutyric-acid (GABA)-releasing (GABAergic) neuronal markers. PDGFRα+ cells were induced from mESC on collagen IV-coated plates in mesenchymal stem cell (MSC) culture medium with limited exposure to retinoic acid, sorted by fluorescence-activated cell sorter and maintained in MSC culture medium containing Y-27632, a Rho-associated kinase inhibitor. We found that supplementation of vascular endothelial growth factor, fibroblast growth factor-basic, and sodium azide (NaN3) to MSC culture medium effectively differentiated PDGFRα+ cells into cells that express GABAergic neuronal markers, such as Pax2, Dlx2, GAD67 NCAM, and tubulin-βIII, while markers for oligodendrocyte (Sox2) and astrocyte (Glast) were suppressed. Immunostaining for GABA showed the majority (86 ± 5%) of the induced cells were GABA-positive. We also found that the PDGFRα+ cells retained such differentiation potential even after more than ten passages and cryopreservation. In summary, this study presents a simple and highly efficient method of inducing cells that express GABAergic neuronal markers from mESC. Together with its ease of maintenance in vitro, PDGFRα+ cells derived from mESC may serve as a useful source for such purpose.

Effect of fluid shear stress on in vitro cultured ureteric bud cells

Most kidney cells are continuously exposed to fluid shear stress (FSS) from either blood flow or urine flow. Recent studies suggest that changes in FSS could contribute to the function and injury of these kidney cells. However, it is unclear whether FSS influences kidney development when urinary flow starts in the embryonic kidneys. In this study, we evaluated the influence of FSS on in vitro cultured ureteric bud (UB) cells by using a pumpless microfluidic device, which offers the convenience of conducting parallel cell culture experiments while also eliminating the need for cumbersome electronic driven equipment and intricate techniques. We first validated the function of the device by both mathematical model and experimental measurements. UB cells dissected from E15.5 mouse embryonic kidneys were cultured in the pumpless microfluidic device and subjected to FSS in the range of 0.4–0.6 dyn mm−2 for 48 h (dynamic). Control UB cells were similarly cultured in the device and maintained under a no-flow condition (static). We found from our present study that the exposure to FSS for up to 48 h led to an increase in mRNA expression levels of UB tip cell marker genes (Wnt11, Ret, Etv4) with a decrease in stalk cell marker genes (Wnt7b, Tacstd2). In further support of the enrichment of UB tip cell population in response to FSS, we also found that exposure to FSS led to a remarkable reduction in the binding of lectin Dolichos Biflorus Agglutinin. In conclusion, results of our present study show that exposure to FSS led to an enrichment in UB tip cell populations, which could contribute to the development and function of the embryonic kidney when urine flow starts at around embryonic age E15.5 in mouse. Since UB tip cells are known to be the proliferative progenitor cells that contribute to the branching morphogenesis of the collecting system in the kidney, our finding could imply an important link between the FSS from the initiation of urine flow and the development and function of the kidney.

Comprehensive analysis of chromatin signature and transcriptome uncovers functional lncRNAs expressed in nephron progenitor cells

Emerging evidence from recent studies has unraveled the roles of long noncoding RNAs (lncRNAs) in the function of various tissues. However, little is known about the roles of lncRNAs in kidney development. In our present study, we aimed to identify functional lncRNAs in one of the three lineages of kidney progenitor cells, i.e., metanephric mesenchymal (MM) cells. We conducted comprehensive analyses of the chromatin signature and transcriptome by RNA-seq and ChIP-seq. We found seventeen lncRNAs that were expressed specifically in MM cells with an active chromatin signature, while remaining silenced in a bivalent chromatin state in non-MM cells. Out of these MM specific lncRNAs, we identified a lncRNA, Gm29418, in a distal enhancer region of Six2, a key regulatory gene of MM cells. We further identified three transcript variants of Gm29418 by Rapid Amplification of cDNA Ends (RACE), and confirmed that the transcription-start-sites (TSSs) of these variants were consistent with the result of Cap Analysis Gene Expression (CAGE). In support of the enhancer-like function of Gm29418 on Six2 expression, we found that knock-down of Gm29418 by two independent anti-sense locked nucleic acid (LNA) phosphorothioate gapmers suppressed Six2 mRNA expression levels in MM cells. We also found that over-expression of Gm29418 led to an increase in Six2 mRNA expression levels in a mouse MM cell line. In conclusion, we identified a lncRNA, Gm29418, in nephron progenitor cells that has an enhancer-like function on a key regulatory gene, Six2.