Injection of hybrid 3D spheroids composed of podocytes, mesenchymal stem cells, and vascular endothelial cells into the renal cortex improves kidney function and replenishes glomerular podocytes

Abstract

Podocytes are highly differentiated epithelial cells that are crucial for maintaining the glomerular filtration barrier in the kidney. Podocyte injury followed by depletion is the major cause of pathological progression of kidney diseases. Although cell therapy has been considered a promising alternative approach to kidney transplantation for the treatment of kidney injury, the resultant therapeutic efficacy in terms of improved renal function is limited, possibly owing to significant loss of engrafted cells. Herein, hybrid three-dimensional (3D) cell spheroids composed of podocytes, mesenchymal stem cells, and vascular endothelial cells were designed to mimic the glomerular microenvironment and as a cell delivery vehicle to replenish the podocyte population by cell transplantation. After creating a native glomerulus-like condition, the expression of multiple genes encoding growth factors and basement membrane factors that are strongly associated with podocyte maturation and functionality was significantly enhanced. Our in vivo results demonstrated that intrarenal transplantation of podocytes in the form of hybrid 3D cell spheroids improved engraftment efficiency and replenished glomerular podocytes. Moreover, the proteinuria of the experimental mice with hypertensive nephropathy was effectively reduced. These data clearly demonstrated the potential of hybrid 3D cell spheroids for repairing injured kidneys.

Keywords: cell spheroids; cell therapy; glomerulus; kidney injury; podocyte.

FIGURE 1

Podocytes (P), mesenchymal stem cells (M), and vascular endothelial cells (E) assemble into hybrid three‐dimensional (3D) cell spheroids (sph.). Representative (a) phase‐contrast photomicrographs and (b) live/dead fluorescence images of the fabricated 3D cell spheroids. Scale bars, 100 μm

FIGURE 2

(a) Confocal Z‐stack images showing the composition of multiple cell types in hybrid 3D cell spheroids. Scale bars, 100 μm. (b) Fluorescence images of podocytes that migrated from the attached spheroids and (c) the distance that the podocytes traveled (n = 24 pooled from three different cell spheroids). Scale bars, 300 μm. Yellow dashed lines indicate 1 mm from the center of the attached cell spheroids. ns, not significant

FIGURE 3

Mutual interactions among cells within 3D spheroids enhance cellular functionality. Expression levels of mouse (a) Synpo and (b) Vegfa and human (c) VEGFA and (d) HGF mRNA in hybrid 3D cell spheroids determined by real‐time quantitative PCR. The results are expressed as the fold change relative to the control podocytes (n = 3). (e) Western blot for laminin, fibronectin (FN) and β‐actin in hybrid 3D cell spheroids and the corresponding quantitative results normalized to β‐actin (n = 3). Data are presented as mean ± SD. All p values were calculated using one‐way ANOVA with the Tukey correction. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001; ns, not significant

FIGURE 4

3D cell spheroids contain abundant extracellular matrix (ECM) proteins and growth factors. Fluorescence images showing (a) the presence of three cell types and (b) the inherent extracellular matrix and growth factors within hybrid 3D cell spheroids. (c) Representative live/dead images of 3D cell spheroids before and after injection through a needle. Scale bars, 100 μm

FIGURE 5

Direct injection into the renal cortex does not result in significant kidney injury. (a) Representative fluorescent images showing the transplanted cells in kidney. Scale bars, 200 μm. (b) Results of hematoxylin and eosin (H&E) staining showing the injection path (indicated by dotted lines). Scale bars, 200 μm (upper panel) or 50 μm (bottom panel). (c) The blood urea nitrogen (BUN) and urine albumin‐creatinine ratio (UACR) of healthy experimental animals at Day 15 after treatment are normalized to preoperative values (n = 3). Data are presented as mean ± SD. The p values were calculated using two‐tailed Student's t‐test. ns, not significant

FIGURE 6

Transplantation of hybrid 3D cell spheroids improves renal function in mice with hypertensive nephropathy. (a) The blood urea nitrogen (BUN) and urine albumin‐creatinine ratio (UACR) of experimental animals that received saline (n = 3) or PME spheroids (n = 6) are normalized to preoperative values. (b) Number of podocytes per glomerulus of the kidney retrieved from the experimental animals. (c) Fluorescence images showing the engraftment of transplanted large T antigen‐positive podocytes. Inset shows a magnified view. Scale bars, 10 μm. All p values were calculated using two‐tailed Student's t‐test. **p < 0.01; ****p < 0.001; ns, not significant