High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells

Amel Dudakovic, Emily Camilleri, Scott M. Riester, Eric A. Lewallen, Sergiy Kvasha, Xiaoyue Chen, Darcie J. Radel, Jarett M. Anderson, Asha A. Nair, Jared M. Evans, Aaron Krych, Jay Smith, David R Deyle, Janet L. Stein, Gary S. Stein, Hee Jeong Im, Simon M. Cool, Jennifer J Westendorf, Sanjeev Kakar, Allan B DietzAndre J van Wijnen

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.

Original languageEnglish (US)
Pages (from-to)1816-1828
Number of pages13
JournalJournal of Cellular Biochemistry
Volume115
Issue number10
DOIs
StatePublished - 2014

Fingerprint

Stem cells
Mesenchymal Stromal Cells
Adipose Tissue
Tissue
Genes
Biomarkers
E2F1 Transcription Factor
Cells
Cellular radio systems
Cyclins
Flow cytometry
Cell proliferation
Platelets
Histones
Chromatin
Extracellular Matrix
Quality control
Cyclin B2
Packaging
Cell Cycle

Keywords

  • ACTA2
  • ADH1B
  • ADIPOGENESIS
  • ADIPOSE-TISSUE DERIVED STROMAL CELLS
  • ASPN
  • CCNB2
  • CCND1
  • CD105
  • CD44
  • CD73
  • CD90
  • CELL CYCLE
  • CHI3L1
  • CHONDROGENESIS
  • CYCLIN
  • E2F1
  • E2F7
  • E2F8
  • ECM2
  • ENG
  • EXTRACELLULAR MATRIX
  • FIBROBLAST
  • FMOD
  • H19
  • HINFP
  • HIST1H3H
  • HIST1H4A
  • HIST2H4A
  • HIST2H4B
  • HISTONE
  • KLF4
  • LINEAGE-COMMITMENT
  • MESENCHYMAL STEM CELL
  • MULTIPOTENT
  • NANOG
  • NES
  • NPAT
  • NT5E
  • OCT4
  • OGN
  • OSTEOGENESIS
  • PLURIPOTENT
  • PODN
  • POU5F1
  • RARRES2
  • SFRP2
  • SFRP4
  • THY1
  • TNNT3
  • WISP2
  • WNT2
  • WNT2A
  • WNT5A
  • WNT5B
  • WNT7B

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells. / Dudakovic, Amel; Camilleri, Emily; Riester, Scott M.; Lewallen, Eric A.; Kvasha, Sergiy; Chen, Xiaoyue; Radel, Darcie J.; Anderson, Jarett M.; Nair, Asha A.; Evans, Jared M.; Krych, Aaron; Smith, Jay; Deyle, David R; Stein, Janet L.; Stein, Gary S.; Im, Hee Jeong; Cool, Simon M.; Westendorf, Jennifer J; Kakar, Sanjeev; Dietz, Allan B; van Wijnen, Andre J.

In: Journal of Cellular Biochemistry, Vol. 115, No. 10, 2014, p. 1816-1828.

Research output: Contribution to journalArticle

Dudakovic, A, Camilleri, E, Riester, SM, Lewallen, EA, Kvasha, S, Chen, X, Radel, DJ, Anderson, JM, Nair, AA, Evans, JM, Krych, A, Smith, J, Deyle, DR, Stein, JL, Stein, GS, Im, HJ, Cool, SM, Westendorf, JJ, Kakar, S, Dietz, AB & van Wijnen, AJ 2014, 'High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells', Journal of Cellular Biochemistry, vol. 115, no. 10, pp. 1816-1828. https://doi.org/10.1002/jcb.24852
Dudakovic, Amel ; Camilleri, Emily ; Riester, Scott M. ; Lewallen, Eric A. ; Kvasha, Sergiy ; Chen, Xiaoyue ; Radel, Darcie J. ; Anderson, Jarett M. ; Nair, Asha A. ; Evans, Jared M. ; Krych, Aaron ; Smith, Jay ; Deyle, David R ; Stein, Janet L. ; Stein, Gary S. ; Im, Hee Jeong ; Cool, Simon M. ; Westendorf, Jennifer J ; Kakar, Sanjeev ; Dietz, Allan B ; van Wijnen, Andre J. / High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells. In: Journal of Cellular Biochemistry. 2014 ; Vol. 115, No. 10. pp. 1816-1828.
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T1 - High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells

AU - Dudakovic, Amel

AU - Camilleri, Emily

AU - Riester, Scott M.

AU - Lewallen, Eric A.

AU - Kvasha, Sergiy

AU - Chen, Xiaoyue

AU - Radel, Darcie J.

AU - Anderson, Jarett M.

AU - Nair, Asha A.

AU - Evans, Jared M.

AU - Krych, Aaron

AU - Smith, Jay

AU - Deyle, David R

AU - Stein, Janet L.

AU - Stein, Gary S.

AU - Im, Hee Jeong

AU - Cool, Simon M.

AU - Westendorf, Jennifer J

AU - Kakar, Sanjeev

AU - Dietz, Allan B

AU - van Wijnen, Andre J

PY - 2014

Y1 - 2014

N2 - Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.

AB - Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.

KW - ACTA2

KW - ADH1B

KW - ADIPOGENESIS

KW - ADIPOSE-TISSUE DERIVED STROMAL CELLS

KW - ASPN

KW - CCNB2

KW - CCND1

KW - CD105

KW - CD44

KW - CD73

KW - CD90

KW - CELL CYCLE

KW - CHI3L1

KW - CHONDROGENESIS

KW - CYCLIN

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KW - E2F7

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KW - NPAT

KW - NT5E

KW - OCT4

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KW - OSTEOGENESIS

KW - PLURIPOTENT

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KW - POU5F1

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KW - SFRP4

KW - THY1

KW - TNNT3

KW - WISP2

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