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. 2013 Feb 1;12(3):442-51.
doi: 10.4161/cc.23308. Epub 2013 Jan 16.

Cyclin-dependent Kinase 4 Signaling Acts as a Molecular Switch Between Syngenic Differentiation and Neural Transdifferentiation in Human Mesenchymal Stem Cells

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Free PMC article

Cyclin-dependent Kinase 4 Signaling Acts as a Molecular Switch Between Syngenic Differentiation and Neural Transdifferentiation in Human Mesenchymal Stem Cells

Janet Lee et al. Cell Cycle. .
Free PMC article

Abstract

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs.

Keywords: cell cycle arrest; cyclin-dependent kinase 4; glial cells; mesenchymal stem/stromal cells; neural cells; neurodegenerative disease; transdifferentiation.

Figures

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Figure 1. Adipose-derived MSCs selectively commit to transdifferentiate into neural cells following CDK inhibition. (A) Phase contrast image of human adipose-derived MSCs (hAD-MSCs). hAD-MSCs were isolated from the fatty portion of liposuction aspirates. (B) hAD-MSCs were labeled with FITC- or PE-coupled antibodies specific for CD34, CD44, CD45, CD49d, CD73 (not shown), CD90 and CD106 antibodies or immunoglobulin isotype control. The surface phenotype was analyzed by FACS. Colored histograms illustrate the control immunoglobulin and open histograms illustrate the specific antibodies, as indicated. Isolated MSCs are positive for the MSC markers (CD44, CD49d and CD90), whereas they are negative for hematopoietic stem cell markers (CD34, CD45 and CD106). Blue-colored shadows represent the isotype control and green represent the staining against each specified antibodies. (C) Adipogenic differentiation resulted in the formation of lipid vacuoles, which were stained with Oil Red O. Osteogenic differentiation showed a highly enriched extracellular matrix stained with von Kossa. Neurogenic differentiation led to a typical neural cell appearance (i.e., a condensed cell body, spherical and refractile), and were immunostained with anti-MAP2 antibody. (D) hAD-MSCs were isolated from six different donors, and characterized as described in (B and C). hAD-MSCs were treated with 10 μM CDK4i for 24 h and digitally imaged. Treatment with CDK4i induced the morphologic neural transdifferentiation with high efficiency. (E) Quantified comparison of cell death and neural transdifferentiation of hAD-MSCs following CDK inhibitor (PurA, 25 μM and CDK4i, 10 μM) treatment. The rate of differentiating hAD-MSCs was randomly counted from 10 different microscopic images (X200) per sample. Averages were obtained from six different MSCs and donors. (F) hAD-MSCs were treated with Purvalanol A (PurA, 25 μM) and CDK4i (10 μM). Cells were harvested, stained with propidium iodide and analyzed by flow cytometry to determine their DNA contents. (G) Phase contrast images of human fetal white matter progenitor/precursor cells (NPCs), human U251 glioblastoma cells and human AD-MSCs. NPCs were cultured in DMEM (NPC) or DMEM supplemented with ITSFn medium (NPC w/ ITSFn). (H) RT-PCR analysis for neural progenitor/precursor cell markers (NCAM, Msi1, and Sox2), a marker for stem cell multi-potency (Nestin) and mesenchymal stem cell surface markers (CD90 and CD105). GAPDH mRNA was utilized as a control.
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Figure 2. Inhibition of CDK4 activity induces neural transdifferentiation in both human adipose- and bone marrow-derived mesenchymal stem/stromal cells. (A) Human AD-MSCs were treated with the control DMSO or inhibitors selective for CDK1 and CDK2 (data not shown) or CDK4, as indicated, and digitally imaged. Treatment with CDK4 inhibitor (CDK4i, Otava Ltd.) induced the morphologic neural transdifferentiation with high efficiency. (B) Time-lapse microscopic image of neural transdifferentiation of hAD-MSCs by CDK4i treatment. Isolated MSCs were infected with a recombinant adenovirus encoding H2B-RFP-fused protein (rAd-H2B-RFP) to visualize DNA, treated with CDK4i and digitally imaged. The arrow indicates the induced neural transdifferentiation of hAD-MSCs. (C) hAD-MSCs were treated with CDK4 inhibitor or control DMSO, while the 24 h post-treatment neural subtype markers, neural precursor (Nestin), neuron markers (β-tubulin III and MAP2) and glial markers (GFAP and NL3) were amplified by RT-PCR. (D) Human bone marrow-derived MSCs (hBM-MSCs) were labeled with FITC- or PE-coupled antibodies specific for CD34, CD44, CD45, CD49d or CD70 antibodies, and immunoglobulin isotype control. Open histograms illustrate the control immunoglobulin, and colored histograms illustrate the specific antibodies, as indicated. Isolated hBM-MSCs were positive for CD44, CD49d and CD70, but negative for CD34 and CD45. hBM-MSCs were cultured for 2–3 wk in the medium for adipogenic or osteogenic differentiation. Differentiation into adipocytes and osteoblasts was confirmed by staining using Oil Red O and alkaline phosphatase, respectively. (E) Time-lapse microscopic image of neural transdifferentiation of hBM-MSCs by CDK4i treatment. Isolated hBM-MSCs were infected with rAd-H2B-RFP, treated with CDK4i and digitally imaged. The arrow indicates the induced neural transdifferentiation of hBM-MSCs. (F) hBM-MSCs were treated with CDK4 inhibitor or control DMSO, while the 24 h post-treatment neural subtype markers, neural precursor (Nestin), neuron markers (β-tubulin III and MAP2) and glial markers (GFAP and NL3) were amplified by RT-PCR.
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Figure 3. Targeted inhibition of CDK4 expression induces the neural transdifferentiation. (A) To deplete the endogenous CDK1, CDK2 and CDK4, ADSCs were transduced with recombinant adenovirus expressing GFP fused-shCDK1 (rAd-GFP-shCDK1), -shCDK2 (rAd-GFP-shCDK2) or -shCDK4 (rAd-GFP-shCDK4), respectively, in conjugation with HP4-PTD. Forty-eight hours post-transduction, cell lysates were prepared and immunoblotted with anti-CDK1, anti-CDK2, anti-CDK4 and anti-actin antibodies. (B) hAD-MSCs were transduced with rAd-GFP [as a control (data not shown)], rAd-GFP-shCDK1, rAd-GFP-shCDK2 or rAd-GFP-shCDK4 and rAd-H2B-RFP to visualize DNA. Transduced hAD-MSCs were cultured in normal culture medium and digitally monitored by time-lapse microscopy. Images were taken at 6 min intervals, showing the false-colored GFP and RFP emissions. The red-colored arrows indicate the neural cells induced by CDK4 depletion. (C) Neural sub-type marker transcripts (Nestin, S100, NL3 and GFAP) were amplified by RT-PCR from hAD-MSCs transduced with rAd-GFP or rAd-GFP-shCDK4. GAPDH mRNA was utilized as an internal control for RT-PCR.
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Figure 4. Inhibition of CDK4 activity reduces the differentiation potential of adipose-derived MSCs into syngenic lineages. (A) hAD-MSCs were treated with CDK4 inhibitor (2 μM, Otava Ltd.) or control DMSO and then cultured in the adipogenic differentiation media. At 2 wk post-treatment, the MSCs showed the formation of lipid vacuoles, which were stained with Oil Red O. (B) hAD-MSCs were treated with CDK4 inhibitor (2 μM and 10 μM) or control DMSO for 24 h. The hAD-MSCs treated with the high concentration of CDK4 inhibitor showed a typical neural cell appearance. (C and D) hAD-MSCs were treated with CDK4 inhibitor (2 μM) or control DMSO and further cultured in the absence or presence of adipogenic differentiation media for 2 wk. The relative rate of adipogenic differentiation, the lipid vacuoles of which were stained with Oil Red O, was measured at OD500 nm (C). Adipogenic marker transcripts (Adiponectin, Lipoprotein lipase and PPARγ2) were amplified by RT-PCR from the MSCs as above. GAPDH mRNA was utilized as an internal control for RT-PCR (D).
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Figure 5. The reversibility between transdifferentiation and dedifferentiation of adipose-derived MSCs by forced regulation of CDK4 activity. (A and C) The hAD-MSCs were transduced with rAd-H2B-RFP by co-treatment with HP4-PTD, and replaced medium 24 h post-infection. Transduced hAD-MSCs were treated with CDK4 inhibitor (CDK4i) for 12 h, and replaced with normal culture medium. This cycle was repeated twice. Time-lapse microscopy images showing the repeated morphologic changes of hAD-MSCs into neural transdifferentiation and dedifferentiation (A). mRNAs were isolated from hAD-MSCs, and neural sub-type markers transcripts were amplified by RT-PCR (B). Protein extracts were prepared from the above hAD-MSCs, and analyzed by immunoblotting using anti-Rb, anti-phospho-Rb S807/811 (phosphorylated-Rb at residues serine 807 and 811), anti-E2F1, anti-Nestin and anti-actin antibodies (C). (D) A schematic model of neural transdifferentiation of mesodermal hMSCs by CDK4 inhibition. The inhibition of CDK4 activity selectively led to transdifferentiation into neural cells, but attenuated the syngenic differentiation of hMSCs.

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