J Korean Soc Transplant.  2015 Sep;29(3):118-129. 10.4285/jkstn.2015.29.3.118.

Retinoic Acid-induced Differentiation of Rat Mesenchymal Stem Cells into beta-Cell Lineage

• Kim JH
• Kim KS
• Lee SW
• Kim HW
• Joo DJ
• Kim YS
• Suh H

• KMID: 2202584
• DOI: http://doi.org/10.4285/jkstn.2015.29.3.118

Abstract

BACKGROUND
S: Type I diabetes mellitus (T1DM), an autoimmune disease, is associated with insulin deficiency due to the death of beta-cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of tissue repair and thus are a promising source of beta-cell surrogates.
METHODS
In this study, the therapeutic potential of BM-MSCs as beta-cell replacements was analyzed both in vitro and in vivo. First, we used retinoic acid (RA) to induce rat BM-MSCs to differentiate into cells of endodermal/pancreatic lineage. Then, differentiated rat BM-MSCs were syngeneically injected under the renal capsule of rats.
RESULTS
Analysis of gene expression revealed that rat BM-MSCs showed signs of early pancreatic development, and differentiated cells were qualitatively and quantitatively confirmed to produce insulin in vitro. In vivo study was performed for short-term (3 weeks) and long-term (8 weeks) period of time. Rats that were injected with differentiated MSCs exhibited a reduction in blood glucose levels throughout 8 weeks, and grafted cells survived in vivo for at least 3 weeks.
CONCLUSIONS
These findings show that RA can induce differentiation of MSCs into the beta-cell lineage and demonstrate the potential of BM-MSCs to serve as therapeutic tools for T1DM.

Keyword

Type 1 diabetes mellitus; Insulin-secreting cells; Mesenchymal stromal cells; Tretinoin; Insulin

MeSH Terms

Animals
Autoimmune Diseases
Blood Glucose
Diabetes Mellitus
Diabetes Mellitus, Type 1
Gene Expression
Insulin
Insulin-Secreting Cells
Mesenchymal Stromal Cells*
Rats*
Transplants
Tretinoin
Blood Glucose
Insulin
Tretinoin

Figure

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  Fig. 1. Fluorescence-activated cell sorting analysis for characterization of rat mesenchymal stem cells (MSCs) at passage 3. Abbreviation: IgG, immunoglobulin G.

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  Fig. 2. Morphological changes of differentiated rat bone marrowderived mesenchymal stem cells. (A, B) Week 1. (C, D) Week 2. (E, F) Week 3 (A, C, E: ×40; B, D, F: ×100).

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  Fig. 3. Reverse-transcriptase polymerase chain reaction of rat bone marrowderived mesenchymal stem cells (rBM-MSCs). Lanes 1 to 3: differentiated rBM-MSCs from week 1 to 3. Abbreviations: FoxA2, forkhead box A2; PDX-1, pancreatic-duodenal homeobox 1; Ngn3, neurogenin 3; Pax4, paired box gene 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 4. Immunocytochemistry of differentiated rat bone marrowderived mesenchymal stem cells at week 3. (A) Insulin (green). (B) 4’, 6-diamidino-2-phenyindole (DAPI, blue). (C) Light microscopic image. (D) Merged insulin and DAPI images. (E) Merged insulin, DAPI, and light microscopic images (magnification, ×100; scale bar=200  m).

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  Fig. 5. Insulin secretion from differentiated rat bone marrowderived mesenchymal stem cells (MSCs) measured by enzyme-linked Immunosorbent Assay at week 3 (n=9). a P<0.05.

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  Fig. 6. Blood glucose levels of rats observed for 3 (A, B) or 8 (C) weeks. (A) Positive control (n=4) and sham control (n=4). (B) Blood glucose level of normal mesenchymal stem cell (MSC)-injected (n=9) and differentiated MSC-injected (n=8) rats. (C) Positive control (n=4) and differentiated MSC-injected (n=8) rats. (D) Linear regression of blood glucose levels from MSC-injected (n=9) and differentiated MSC-injected (n=8) rats. Slope for MSC-injected group=−0.4935±0.5103; slope for differentiated MSC-injected group=−1.728±0.7725.

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  Fig. 7. 3,3’-Diaminobenzidine-stained tissue. (A) Cells located along the inner surface of the tissue membrane. (B) Cell that migrated a short distance into the tissue (marked by yellow arrowhead) (magnification: ×100; scale bar=200 m).

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  Fig. 8. PKH26-labeled cells in kidney capsule (marked by yellow arrowheads). (A, B) Cell located along the inner surface of the tissue membrane. (C) Cells that migrated a short distance into the tissue (magnification: ×100; scale bar=200  m).

Reference

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