In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications

L. Roseti, M. Serra, F. Canella, C. Munno, A. Tosi, M. Zuntini, M. Pandolfi, L. Sangiorgi, P. Biso, M. C. Pittalis, C. Bini, S. Pelotti, A. Gasbarrini, L. Boriani, A. Bassi, B. Grigolo

Research output: Contribution to journalArticle

Abstract

OBJECTIVE: A number of studies have shown the role of expanded Bone Marrowderived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications L. ROSETI, M. SERRA, F. CANELLA1, C. MUNNO1, A. TOSI1, M. ZUNTINI2, M. PANDOLFI2, L. SANGIORGI2, P. BISO3, M.C. PITTALIS3, C. BINI4, S. PELOTTI4, A. GASBARRINI5, L. BORIANI6, A. BASSI1, B. GRIGOLO7 RAMSES Laboratory, Research Innovation Technology Department, Rizzoli Orthopaedic Institute, Bologna, Italy 1Cell Factory, Prometeo, Research Innovation Technology Department, Rizzoli Orthopaedic Institute, Bologna, Italy 2Department of Medical Genetics and Skeletal Rare Diseases, Rizzoli Orthopaedic Institute, Bologna, Italy 3Department of Medicine and Surgery (DIMEC), Obstetrics and Gynecology, University of Bologna, Bologna, Italy 4Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, Bologna, Italy 5Department of Oncologic and Degenerative Spine Surgery, Rizzoli Orthopaedic Institute, Bologna, Italy 6Orthopaedics and Traumatology Operative Unit, Regional Spinal Department, Ospedale "Alto Vicentino", Santorso (Schio-Thiene, Vicenza), Italy 7Laboratory of Immunorheumatology and Tissue Regeneration, Rizzoli Orthopaedic Institute, Bologna, Italy Corresponding Author: Livia Roseti, Ph.D; e-mail: livia.roseti@ior.it p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10% limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were undetectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes.

Original languageEnglish
Pages (from-to)3702-3711
Number of pages10
JournalEuropean Review for Medical and Pharmacological Sciences
Volume18
Issue number23
Publication statusPublished - 2014

Fingerprint

Mesenchymal Stromal Cells
Italy
Chromosomes
Bone Marrow
Orthopedics
Genes
Karyotyping
DNA Fingerprinting
Genomic Instability
Chromosome Aberrations
Regeneration
Molecular Biology
Obstetric Surgical Procedures
Medicine
Technology
Gene Expression
Traumatology
Forensic Medicine
In Vitro Techniques
Medical Genetics

Keywords

  • Direct sequencing
  • DNA profiling
  • Karyotyping
  • Mesenchymal stem cells
  • Musculo-skeletal applications
  • Oncogenes
  • Tumor suppressor genes

ASJC Scopus subject areas

  • Medicine(all)
  • Pharmacology (medical)

Cite this

In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications. / Roseti, L.; Serra, M.; Canella, F.; Munno, C.; Tosi, A.; Zuntini, M.; Pandolfi, M.; Sangiorgi, L.; Biso, P.; Pittalis, M. C.; Bini, C.; Pelotti, S.; Gasbarrini, A.; Boriani, L.; Bassi, A.; Grigolo, B.

In: European Review for Medical and Pharmacological Sciences, Vol. 18, No. 23, 2014, p. 3702-3711.

Research output: Contribution to journalArticle

Roseti, L, Serra, M, Canella, F, Munno, C, Tosi, A, Zuntini, M, Pandolfi, M, Sangiorgi, L, Biso, P, Pittalis, MC, Bini, C, Pelotti, S, Gasbarrini, A, Boriani, L, Bassi, A & Grigolo, B 2014, 'In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications', European Review for Medical and Pharmacological Sciences, vol. 18, no. 23, pp. 3702-3711.
Roseti, L. ; Serra, M. ; Canella, F. ; Munno, C. ; Tosi, A. ; Zuntini, M. ; Pandolfi, M. ; Sangiorgi, L. ; Biso, P. ; Pittalis, M. C. ; Bini, C. ; Pelotti, S. ; Gasbarrini, A. ; Boriani, L. ; Bassi, A. ; Grigolo, B. / In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications. In: European Review for Medical and Pharmacological Sciences. 2014 ; Vol. 18, No. 23. pp. 3702-3711.
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abstract = "OBJECTIVE: A number of studies have shown the role of expanded Bone Marrowderived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications L. ROSETI, M. SERRA, F. CANELLA1, C. MUNNO1, A. TOSI1, M. ZUNTINI2, M. PANDOLFI2, L. SANGIORGI2, P. BISO3, M.C. PITTALIS3, C. BINI4, S. PELOTTI4, A. GASBARRINI5, L. BORIANI6, A. BASSI1, B. GRIGOLO7 RAMSES Laboratory, Research Innovation Technology Department, Rizzoli Orthopaedic Institute, Bologna, Italy 1Cell Factory, Prometeo, Research Innovation Technology Department, Rizzoli Orthopaedic Institute, Bologna, Italy 2Department of Medical Genetics and Skeletal Rare Diseases, Rizzoli Orthopaedic Institute, Bologna, Italy 3Department of Medicine and Surgery (DIMEC), Obstetrics and Gynecology, University of Bologna, Bologna, Italy 4Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, Bologna, Italy 5Department of Oncologic and Degenerative Spine Surgery, Rizzoli Orthopaedic Institute, Bologna, Italy 6Orthopaedics and Traumatology Operative Unit, Regional Spinal Department, Ospedale {"}Alto Vicentino{"}, Santorso (Schio-Thiene, Vicenza), Italy 7Laboratory of Immunorheumatology and Tissue Regeneration, Rizzoli Orthopaedic Institute, Bologna, Italy Corresponding Author: Livia Roseti, Ph.D; e-mail: livia.roseti@ior.it p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10{\%} limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were undetectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes.",
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T1 - In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications

AU - Roseti, L.

AU - Serra, M.

AU - Canella, F.

AU - Munno, C.

AU - Tosi, A.

AU - Zuntini, M.

AU - Pandolfi, M.

AU - Sangiorgi, L.

AU - Biso, P.

AU - Pittalis, M. C.

AU - Bini, C.

AU - Pelotti, S.

AU - Gasbarrini, A.

AU - Boriani, L.

AU - Bassi, A.

AU - Grigolo, B.

PY - 2014

Y1 - 2014

N2 - OBJECTIVE: A number of studies have shown the role of expanded Bone Marrowderived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications L. ROSETI, M. SERRA, F. CANELLA1, C. MUNNO1, A. TOSI1, M. ZUNTINI2, M. PANDOLFI2, L. SANGIORGI2, P. BISO3, M.C. PITTALIS3, C. BINI4, S. PELOTTI4, A. GASBARRINI5, L. BORIANI6, A. BASSI1, B. GRIGOLO7 RAMSES Laboratory, Research Innovation Technology Department, Rizzoli Orthopaedic Institute, Bologna, Italy 1Cell Factory, Prometeo, Research Innovation Technology Department, Rizzoli Orthopaedic Institute, Bologna, Italy 2Department of Medical Genetics and Skeletal Rare Diseases, Rizzoli Orthopaedic Institute, Bologna, Italy 3Department of Medicine and Surgery (DIMEC), Obstetrics and Gynecology, University of Bologna, Bologna, Italy 4Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, Bologna, Italy 5Department of Oncologic and Degenerative Spine Surgery, Rizzoli Orthopaedic Institute, Bologna, Italy 6Orthopaedics and Traumatology Operative Unit, Regional Spinal Department, Ospedale "Alto Vicentino", Santorso (Schio-Thiene, Vicenza), Italy 7Laboratory of Immunorheumatology and Tissue Regeneration, Rizzoli Orthopaedic Institute, Bologna, Italy Corresponding Author: Livia Roseti, Ph.D; e-mail: livia.roseti@ior.it p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10% limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were undetectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes.

AB - OBJECTIVE: A number of studies have shown the role of expanded Bone Marrowderived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications L. ROSETI, M. SERRA, F. CANELLA1, C. MUNNO1, A. TOSI1, M. ZUNTINI2, M. PANDOLFI2, L. SANGIORGI2, P. BISO3, M.C. PITTALIS3, C. BINI4, S. PELOTTI4, A. GASBARRINI5, L. BORIANI6, A. BASSI1, B. GRIGOLO7 RAMSES Laboratory, Research Innovation Technology Department, Rizzoli Orthopaedic Institute, Bologna, Italy 1Cell Factory, Prometeo, Research Innovation Technology Department, Rizzoli Orthopaedic Institute, Bologna, Italy 2Department of Medical Genetics and Skeletal Rare Diseases, Rizzoli Orthopaedic Institute, Bologna, Italy 3Department of Medicine and Surgery (DIMEC), Obstetrics and Gynecology, University of Bologna, Bologna, Italy 4Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, Bologna, Italy 5Department of Oncologic and Degenerative Spine Surgery, Rizzoli Orthopaedic Institute, Bologna, Italy 6Orthopaedics and Traumatology Operative Unit, Regional Spinal Department, Ospedale "Alto Vicentino", Santorso (Schio-Thiene, Vicenza), Italy 7Laboratory of Immunorheumatology and Tissue Regeneration, Rizzoli Orthopaedic Institute, Bologna, Italy Corresponding Author: Livia Roseti, Ph.D; e-mail: livia.roseti@ior.it p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10% limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were undetectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes.

KW - Direct sequencing

KW - DNA profiling

KW - Karyotyping

KW - Mesenchymal stem cells

KW - Musculo-skeletal applications

KW - Oncogenes

KW - Tumor suppressor genes

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