WNT and VEGF/PDGF signaling regulate self-renewal in primitive mesenchymal stem cells

Background Therapeutic use of multipotent mesenchymal stem cells (MSCs) is hampered due to poor growth and limited self-renewal potential. The self-renewal potential of MSCs is also affected during propagation and changes are poorly understood. This study investigated the molecular mechanism involved in the self-renewal of primitive (p) MSCs. Methods pMSCs were cultured to low passage (LP), P3, and high passage (HP), P20, in fetal bovine serum medium (FM) and xeno-free medium (XM). The characteristics of LP and HP pMSCs were evaluated for morphology, expression of cell surface markers, doubling time (DT), colony forming efficiency (CFE), proliferation by BrdU assay, telomerase activity and trilineage differentiation. We then examined transcriptome and nucleosome occupancies using RNA-seq and MNase-seq, respectively analyses. Results pMSCs grown in FM gradually changed morphology to large elongated cells and showed a significant reduction in the expression of CD90 and CD49f, CFE, proliferation, and telomerase activity. In addition, cells had a greater propensity to differentiate into the adipogenic lineage. In contrast, pMSCs grown in XM maintained small fibroblastoid morphology, self-renewal, and differentiation potential. Transcriptomic analysis showed upregulation of genes involved in self-renewal, cell cycle, and DNA replication in XM-grown pMSCs. Whereas senescence genes were upregulated in cells in FM. MNase-seq analysis revealed less nucleosomal occupancies in self-renewal genes and senescence genes in pMSCs grown in XM and FM, respectively. The expression of selected genes associated with self-renewal, cell cycle, DNA replication, differentiation, and senescence was confirmed by qRT-PCR. These results led us to propose signaling pathways involved in the self-renewal and senescence of pMSCs. Conclusion We conclude that the self-renewal potential of pMSCs is controlled by WNT and VEGF/PDGF, but TGFβ and PI3K signaling induce senescence.

[1]  K. Yarygin,et al.  Telomeres and Telomerase in the Control of Stem Cells , 2022, Biomedicines.

[2]  G. Chaudhry,et al.  The potential role of integrin alpha 6 in human mesenchymal stem cells , 2022, Frontiers in Genetics.

[3]  G. Chaudhry,et al.  Human primitive mesenchymal stem cell-derived retinal progenitor cells improved neuroprotection, neurogenesis, and vision in rd12 mouse model of retinitis pigmentosa , 2022, Stem cell research & therapy.

[4]  Gang Yin,et al.  Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities , 2022, Signal Transduction and Targeted Therapy.

[5]  D. Feinstein,et al.  Neural stem cells derived from primitive mesenchymal stem cells reversed disease symptoms and promoted neurogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis , 2021, Stem Cell Research & Therapy.

[6]  Jae-Ho Kim,et al.  Enhancement of anti-inflammatory and immunomodulatory effects of adipose-derived human mesenchymal stem cells by making uniform spheroid on the new nano-patterned plates. , 2021, Biochemical and biophysical research communications.

[7]  M. Janowski,et al.  Immunomodulatory and Regenerative Effects of Mesenchymal Stem Cells and Extracellular Vesicles: Therapeutic Outlook for Inflammatory and Degenerative Diseases , 2021, Frontiers in Immunology.

[8]  P. Vogt,et al.  Comparative Analysis of Mesenchymal Stem Cell Cultivation in Fetal Calf Serum, Human Serum, and Platelet Lysate in 2D and 3D Systems , 2021, Frontiers in Bioengineering and Biotechnology.

[9]  Maite Huarte,et al.  Gene regulation by long non-coding RNAs and its biological functions , 2020, Nature reviews. Molecular cell biology.

[10]  H. T. Bui,et al.  Influences of Xeno-Free Media on Mesenchymal Stem Cell Expansion for Clinical Application , 2020, Tissue Engineering and Regenerative Medicine.

[11]  C. Govind,et al.  Transcriptomic Analysis of Naïve Human Embryonic Stem Cells Cultured in Three-Dimensional PEG Scaffolds , 2020, bioRxiv.

[12]  P. Vogt,et al.  Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues. , 2020, Cytotherapy.

[13]  C. Govind,et al.  Mesenchymal stem cells: Cell therapy and regeneration potential , 2019, Journal of tissue engineering and regenerative medicine.

[14]  G. Rho,et al.  Differentiation Inductions Altered Telomere Length and Telomerase Activity in Human Dental PulpDerived Mesenchymal Stem Cell , 2019, Journal of Animal Reproduction and Biotechnology.

[15]  T. Reichert,et al.  Short telomeres correlate with a strong induction of cellular senescence in human dental follicle cells , 2019, BMC Molecular and Cell Biology.

[16]  M. Aljamali,et al.  Isolation of umbilical cord mesenchymal stem cells using human blood derivatives accompanied with explant method. , 2019, Stem cell investigation.

[17]  Yizhi Liu,et al.  Wnt5a Contributes to the Differentiation of Human Embryonic Stem Cells into Lentoid Bodies Through the Noncanonical Wnt/JNK Signaling Pathway. , 2018, Investigative ophthalmology & visual science.

[18]  Daniel J. Blankenberg,et al.  The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update , 2018, Nucleic Acids Res..

[19]  G. Barabino,et al.  Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro , 2018, Stem Cell Research & Therapy.

[20]  F. Iqbal,et al.  In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells. , 2017, Journal of visualized experiments : JoVE.

[21]  P. Krebsbach,et al.  The Role of Integrin α6 (CD49f) in Stem Cells: More than a Conserved Biomarker. , 2017, Stem cells and development.

[22]  Y. Shin,et al.  Enhanced Cell Growth of Adipocyte-Derived Mesenchymal Stem Cells Using Chemically-Defined Serum-Free Media , 2017, International journal of molecular sciences.

[23]  G. Chaudhry,et al.  Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta , 2017, Journal of visualized experiments : JoVE.

[24]  J. Cook,et al.  Cell cycle proliferation decisions: the impact of single cell analyses , 2017, The FEBS journal.

[25]  L. Martinez,et al.  Mitochondrial Functional Changes Characterization in Young and Senescent Human Adipose Derived MSCs , 2016, Front. Aging Neurosci..

[26]  D. Bhavanasi,et al.  Wnt Signaling in Normal and Malignant Stem Cells , 2016, Current Stem Cell Reports.

[27]  J. Savickienė,et al.  Senescence-Associated Molecular and Epigenetic Alterations in Mesenchymal Stem Cell Cultures from Amniotic Fluid of Normal and Fetus-Affected Pregnancy , 2016, Stem cells international.

[28]  A. Pic-Taylor,et al.  A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells , 2016, Stem Cell Research & Therapy.

[29]  G. Wilson,et al.  Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord. , 2016, Stem cell research.

[30]  E. Hwang,et al.  Chemical and Physical Approaches to Extend the Replicative and Differentiation Potential of Stem Cells , 2016, Stem Cell Reviews and Reports.

[31]  Yuanzhi Xu,et al.  Cross-Talking Between PPAR and WNT Signaling and its Regulation in Mesenchymal Stem Cell Differentiation. , 2016, Current stem cell research & therapy.

[32]  N. Wlodarchak,et al.  PP2A as a master regulator of the cell cycle , 2016, Critical reviews in biochemistry and molecular biology.

[33]  H. Liber,et al.  Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis , 2015, Front. Oncol..

[34]  Alyssa Ziman,et al.  Optimization of human mesenchymal stem cell manufacturing: the effects of animal/xeno-free media , 2015, Scientific Reports.

[35]  O. Klezovitch,et al.  Cadherin signaling: keeping cells in touch , 2015, F1000Research.

[36]  D. Gomes,et al.  Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation , 2015, Stem Cell Research & Therapy.

[37]  J. Nevins,et al.  Division of labour between Myc and G1 cyclins in cell cycle commitment and pace control , 2014, Nature Communications.

[38]  P. Venugopal,et al.  Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton’s jelly derived mesenchymal stem cells? A comparative study , 2014, Stem Cell Research & Therapy.

[39]  Manuel Serrano,et al.  Cellular senescence: from physiology to pathology , 2014, Nature Reviews Molecular Cell Biology.

[40]  K. Lyons,et al.  TGFβ signaling in cartilage development and maintenance. , 2014, Birth defects research. Part C, Embryo today : reviews.

[41]  Udaykumar Kolkundkar,et al.  Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions , 2014, Cell and Tissue Research.

[42]  A. Dopazo,et al.  Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy , 2013, Cell Death and Disease.

[43]  P. Pinton,et al.  Donor Age-Related Biological Properties of Human Dental Pulp Stem Cells Change in Nanostructured Scaffolds , 2012, PloS one.

[44]  Kyung Yong Lee,et al.  Human umbilical cord blood-derived mesenchymal stem cells undergo cellular senescence in response to oxidative stress. , 2012, Stem cells and development.

[45]  X. Graña,et al.  PP2A holoenzymes negatively and positively regulate cell cycle progression by dephosphorylating pocket proteins and multiple CDK substrates. , 2012, Gene.

[46]  F. D. D. Fagagna,et al.  Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation , 2012, Nature Cell Biology.

[47]  Fei Huang,et al.  Regulation of TGF-β receptor activity , 2012, Cell & Bioscience.

[48]  W. Vermeulen,et al.  ATP-dependent chromatin remodeling in the DNA-damage response , 2012, Epigenetics & Chromatin.

[49]  Yaojiong Wu,et al.  Epigenetic Dysregulation in Mesenchymal Stem Cell Aging and Spontaneous Differentiation , 2011, PLoS ONE.

[50]  K. Kang,et al.  DNA Methyltransferase Controls Stem Cell Aging by Regulating BMI1 and EZH2 through MicroRNAs , 2011, PloS one.

[51]  Li-Rong Yu,et al.  Distinct roles of GCN5/PCAF‐mediated H3K9ac and CBP/p300‐mediated H3K18/27ac in nuclear receptor transactivation , 2011, The EMBO journal.

[52]  Wolfgang Wagner,et al.  Different facets of aging in human mesenchymal stem cells. , 2010, Tissue engineering. Part B, Reviews.

[53]  Zhiwei Wang,et al.  Emerging role of Notch in stem cells and cancer. , 2009, Cancer letters.

[54]  B. Lo,et al.  Ethical issues in stem cell research. , 2009, Endocrine reviews.

[55]  Seong Who Kim,et al.  Down-regulation of CD105 is associated with multi-lineage differentiation in human umbilical cord blood-derived mesenchymal stem cells. , 2009, Biochemical and biophysical research communications.

[56]  J. Nevins,et al.  A role for Myc in facilitating transcription activation by E2F1 , 2008, Oncogene.

[57]  F. D. D. Fagagna.  Living on a break: cellular senescence as a DNA-damage response , 2008, Nature Reviews Cancer.

[58]  A. Cometa,et al.  Human bone marrow-derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms , 2010 .

[59]  J. Campisi,et al.  Cellular senescence: when bad things happen to good cells , 2007, Nature Reviews Molecular Cell Biology.

[60]  J. Shay,et al.  Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol , 2006, Nature Protocols.

[61]  I. Matsumura,et al.  Roles for c-Myc in Self-renewal of Hematopoietic Stem Cells* , 2004, Journal of Biological Chemistry.

[62]  G Gstraunthaler,et al.  The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. , 2004, Toxicology in vitro : an international journal published in association with BIBRA.

[63]  D. Eick,et al.  Myc/Max/Mad regulate the frequency but not the duration of productive cell cycles , 2001, EMBO reports.

[64]  M E Greenberg,et al.  Myc requires distinct E2F activities to induce S phase and apoptosis. , 2001, Molecular cell.

[65]  M. Björkholm,et al.  Real-time quantitative telomeric repeat amplification protocol assay for the detection of telomerase activity. , 2001, Clinical chemistry.

[66]  J. Shay,et al.  Lack of cell cycle regulation of telomerase activity in human cells. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[67]  G. Gurtner,et al.  Mesenchymal Stem Cells Home to Sites of Injury and Inflammation , 2012 .

[68]  D. Prockop,et al.  Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. , 2006, Cytotherapy.