Hyung Kim Jae; Sik Kim Kyung; Woo Lee Sang; Woo Kim Hyun; Jin Joo Dong; Seun Kim Yu; Hwal Suh

doi:10.4285/jkstn.2015.29.3.118

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Jae, Kyung, Sang, Hyun, Dong, Yu, and Suh: Retinoic Acid-induced Differentiation of Rat Mesenchymal Stem Cells into -Cell Lineage

Original Article

J Korean Soc Transplant 2015;29(3):118-129.

Published online: 10 September 2015

DOI: https://doi.org/10.4285/jkstn.2015.29.3.118

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

Hyung Kim Jae, Ph.D.^1,², Sik Kim Kyung, M.D.^1,³, Woo Lee Sang, B.S.^1,², Woo Kim Hyun, M.D.^1,⁴, Jin Joo Dong, M.D.^3,⁵, Seun Kim Yu, M.D.^1,^3,⁵, Hwal Suh, D.D.S.^1,²

¹Graduate Program of Nano Science and Technology, Graduate School of Yonsei University, Seoul, Korea

²Departments of Medical Engineering, Seoul, Korea

³Surgery, Seoul, Korea

⁴Orthopedic Surgery, Seoul, Korea

⁵Yonsei University College of Medicine, Department of Transplantation Surgery, Severance Hospital, Yonsei University Health System, Seoul, Korea, Seoul, Korea

Corresponding authors: Yu Seun Kim, Department of Transplantation Surgery, Severance Hospital, Yonsei University Health System, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea Tel: 82-2-2228-2115, Fax: 82-2-313-8289 E-mail: yukim@yuhs.ac

Hwal Suh, Department of Medical Engineering, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea Tel: 82-2-2228-1918, Fax: 82-2-363-9923 E-mail: hwal@yuhs.ac This work was mainly performed by the laboratory expense of Department of Medical Engineering, Yonsei University College of Medicine. It was supported in part, to purchase reagents and animals, by Yonsei University (http://research.yonsei.ac.kr) IACF (2013-31-0834).

Received 13 June 2015 Revised 6 July 2015 Accepted 6 July 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Backgrounds

Type I diabetes mellitus (T1DM), an autoimmune disease, is associated with insulin deficiency due to the death of -cells. Bone marrowderived mesenchymal stem cells (BM-MSCs) are capable of tissue repair and thus are a promising source of -cell surrogates.

Methods

In this study, the therapeutic potential of BM-MSCs as -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 -cell lineage and demonstrate the potential of BM-MSCs to serve as therapeutic tools for T1DM.

Keywords: Type 1 diabetes mellitus, Insulin-secreting cells, Mesenchymal stromal cells, Tretinoin, Insulin

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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.

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).

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.

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).

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.

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.

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).

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).

Table 1.

Primers for rat bone marrowderived mesenchymal stem cells

Gene	Sequence	Accession number	Product size (bp)	Annealing Temperature (^o C)	Cycles
FoxA2	Sense: 5’-AAG GGA AAT GAC AGG CTG AGT GGA-3’ Antisense: 5’-TGT GGA ACT CTG GCA TTC TAG CCA-3’	NM_012743.1	581	63	30
PDX-1	Sense: 5’-TGC CAC CAT GAA TAG TGA GGA GCA-3’ Antisense: 5’-CGC GTG AGC TTT GGT GGA TTT CAT-3’	NM_022852.3	394	63	30
Ngn3	Sense: 5’-TTA CAA AGA TCG AGA CCC TGC GCT-3’ Antisense: 5’-AAG AGC CAG TGA GGT AAG ACG CAA-3’	NM_021700.1	670	63	30
Pax4	Sense: 5’-AGC TGA GGC ACT GGA GAA AGA GTT-3’ Antisense: 5’-AGC ACA GCT GAC AGA AGG AAG GAT-3’	NM_031799.1	305	63	30
C-peptide	Sense: 5’-TGT CAA ACA GCA CCT TTG TGG TCC-3’ Antisense: 5’-AGT GGT GGA CTC AGT TGC AGT AGT-3’	NM_019129.3	500	63	30

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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