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miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells

Abstract

This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.

Abbreviations

α-MEM:

alpha minimal essential medium

BMSCs:

bone marrow stem cells

BSA:

bovine serum albumin

CD34:

cluster of differentiation 34

CD44:

cluster of differentiation 44

CD90:

cluster of differentiation 90

CD105:

cluster of differentiation 105

CD133:

cluster of differentiation 133

cDNA:

complementary DNA

Ct:

cycle threshold

DMEM:

Dulbecco’s modified Eagle’s medium

DMEM F12:

Dulbecco’s modified Eagle’s medium: nutrient mixture F-12

DTZ:

diphenylthiocarbazone

ESC:

embryonic stem cell

FACS:

fluorescence-activated cell sorting

FAM:

6-carboxyfluorescein

GATA6:

transcription factor GATA-6

(GLP)-1:

glucagon-like peptide-1

GLUT2:

glucose transporter 2

GVHD:

graft-versus-host disease

HBV:

hepatitis B virus

HCV:

hepatitis C virus

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HIV:

human immunodeficiency virus

Hnf1β:

hepatocyte nuclear factor1β

HNF6:

hepatocyte nuclear factor 6

hPDMSCs:

human placenta-derived mesenchymal stem cells

ICC:

immunocytochemistry

IPCs:

insulin-producing clusters

ITS:

insulin-transferrin-selenium

KIR6. 2:

potassium inwardly-rectifying channel

KRH buffer:

Krebs’ ringer bicarbonate HEPES buffer

miR:

micro ribonucleic acid

miRNAs:

micro ribonucleic acid

MSCs:

mesenchymal stem cells

NEUROD1:

neuronal differentiation 1

NGN3:

neurogenin 3

Nkx6. 1:

NK homeobox factor 6. 1

OCT4:

octamer binding transcription factor 4 (transcription factor expressed by embryonic stem cells)

Opti-MEM:

Opti-Minimum Essential Media

PAX4:

paired homeobox transcription factor 4

PAX6:

paired homeobox transcription factor 6

PBS:

phosphate buffered saline

PDB-MSCs:

human placental decidua basalismesenchymal stem cells

PDX-1:

pancreatic and duodenal homeobox 1

Sox17:

SRY-related HMG box 17

SVF:

stromal-vascular cell fraction

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Correspondence to Negar Azarpira.

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Shaer, A., Azarpira, N., Vahdati, A. et al. miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells. Cell Mol Biol Lett 19, 483–499 (2014). https://doi.org/10.2478/s11658-014-0207-3

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  • DOI: https://doi.org/10.2478/s11658-014-0207-3

Keywords

  • Pancreas
  • Beta cells
  • miR-375
  • Placenta
  • Mesenchymal stromal cells
  • Induced pluripotent stem cell
  • microRNA
  • Insulin
  • Differentiation
  • NGN3
  • GLUT2
  • PDX1