Combined Placental Mesenchymal Stem Cells with Guided Nanoparticles Effective Against Diabetic Nephropathy in Mouse Model

Abstract

Background: Diabetic nephropathy (DN) is a prevalent complication of diabetes mellitus and constitutes the primary cause of mortality in affected patients. Previous studies have shown that placental mesenchymal stem cells (PL-MSCs) can alleviate kidney dysfunction in animal models of DN. However, the limited ability of mesenchymal stem cells (MSCs) to home to damaged sites restricts their therapeutic potential. Enhancing the precision of PL-MSCs' homing to target tissues is therefore vital for the success of cell therapies in treating DN.

Methods: We developed Fe₃O₄ coated polydopamine nanoparticle (NP)-internalized MSCs and evaluated their therapeutic effectiveness in a mouse model of streptozotocin- and high-fat diet-induced DN, using an external magnetic field.

Results: Our study confirmed that NPs were effectively internalized into PL-MSCs without compromising their intrinsic stem cell properties. The magnetic targeting of PL-MSCs notably improved their homing to the kidney tissues in mice with DN, resulting in enhanced kidney function compared to the transplantation of PL-MSCs alone. Furthermore, the anti-inflammatory and antifibrotic attributes of PL-MSCs played a role in the recovery of kidney function and structure.

Conclusion: These results demonstrate that magnetically targeted therapy using PL-MSCs is a promising approach for treating diabetic nephropathy.

Keywords: diabetic nephropathy; iron oxide nanoparticles; magnetic targeting; mesenchymal stem cells.

Figure 1

Schematic Overview of the Entire Experiment.

Figure 2

Internalization of Nanoparticles (NPs) into Placental Mesenchymal Stem Cells (PL-MSCs). (A) MSCs stained with Prussian Blue to highlight iron (Fe) content. Scale bar = 50 µm. (B) Quantification of Fe concentration using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) (n=3). (C) Assessment of NP-induced cytotoxicity in MSCs labeled with NPs at varying concentrations for 24 h (n=4). (D) Measurement of β-galactosidase levels in NP-labeled MSCs using ELISA (n=4). (E) Proliferation rates of cells labeled with NPs at different concentrations (n=5). (F and G) Transmission Electron Microscopy (TEM) images showing NPs internalized into an MSC at a concentration of 50 µg/mL. The red arrow indicates NP. Data are presented as mean ± S.E.M. P-values < 0.05 were considered statistically significant, denoted as *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Fundamental Characteristics of Nanoparticle (NP)-Labeled Placental Mesenchymal Stem Cells (PL-MSCs). (A) Immunophenotyping of MSCs labeled with NPs compared to unlabeled MSCs. (B) Differentiation potential of MSCs into adipogenic and osteogenic lineages under various culture conditions.

Figure 4

Comparative Effects of NP-Labeled and Unlabeled Placental Mesenchymal Stem Cell (PL-MSC) Treatments on Biochemical Indices in Mice with Diabetic Nephropathy (DN). (A) 24-hour water intake (n=5), (B) Body weight (n=5), (C) Blood glucose concentration curve (n=5), (D) Kidney weight (n=5), (E) Kidney index (n=5), (F) Serum urea nitrogen (n=5), (G) Serum creatinine (n=5), (H) Urine creatinine (n=5), and (I) Urinary albumin (n=5). Data are presented as mean ± S.E.M. P-values < 0.05 were considered statistically significant, denoted as *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Histopathological Analysis of Tissues from Mice with Diabetic Nephropathy (DN). (A) Hematoxylin and Eosin (H&E) staining. (B) Periodic Acid-Schiff (PAS) staining. The red arrow indicates Bowman’s space. (C) Masson’s Trichrome staining. (D) Transmission Electron Microscopy (TEM) of renal tissues. The red arrow indicates thickening of the glomerular basement membrane. (E) Identification and distribution of Placental Mesenchymal Stem Cells (PL-MSCs) in the kidneys of mice with DN. The red arrow indicates MAB1281-positive cells in the kidney tissue.

Figure 6

Impact of Mesenchymal Stem Cells (MSCs) on Inflammatory Cytokines in Mice with Diabetic Nephropathy (DN). (A) Temporal profile of cytokine levels in the serum of DN mice treated with phosphate-buffered saline (PBS), MSCs, nanoparticle (NP)-labeled MSCs, or magnetically targeted PL-MSCs (n=5). (B) Relative mRNA expression of inflammatory cytokines in kidney tissues (n=4). Data are presented as mean ± S.E.M. P-values < 0.05 were considered statistically significant, indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Inflammation in Kidneys of Mice with Diabetic Nephropathy (DN). Immunofluorescence staining is used to identify Ly6G+ neutrophils (A), Scale bar = 100 μm), F4/80+ macrophages (B), Scale bar = 100 μm), and CD3+ lymphocytes (C), Scale bar = 50 μm) in kidney tissue sections. “G” denotes the glomerulus.

Figure 8

Impact of Nanoparticle (NP)-Labeled and Unlabeled Mesenchymal Stem Cell (MSC) Treatments on Kidney Fibrosis in Mice with Diabetic Nephropathy (DN). (A) Immunohistochemistry staining for (B) α-smooth muscle actin and (C) transforming growth factor-β (TGF-β) in kidney tissues. (D) Quantitative analysis of TGF-β mRNA expression in kidney tissues (n=4). (E) Plasma levels of TGF-β (n=5). Data are represented as mean ± S.E.M. P-values < 0.05 were considered statistically significant, indicated as **P < 0.01; ***P < 0.001.