Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration
Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isola...
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| creator | Cuthbert, Richard J Giannoudis, Peter V Wang, Xiao N Nicholson, Lindsay Pawson, David Lubenko, Anatole Tan, Hiang B Dickinson, Anne McGonagle, Dennis Jones, Elena |
| description | Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells.
MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair.
Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs.
A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications. |
| doi_str_mv | 10.1371/journal.pone.0117855 |
| format | Article |
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MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair.
Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs.
A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0117855</identifier><identifier>PMID: 25760857</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adolescent ; Adult ; Aged ; Arthritis ; Autografts ; Bone growth ; Bone healing ; Bone marrow ; Bone morphogenetic protein 2 ; Bone Regeneration ; CD105 antigen ; CD45 antigen ; CD73 antigen ; CD90 antigen ; Cell adhesion & migration ; Cell culture ; Cell Culture Techniques ; Cell Proliferation ; Child ; Child, Preschool ; Clinical trials ; CXCL12 protein ; Cytometry ; Enrichment ; Ethics ; Feasibility studies ; Femur ; Femur Head - cytology ; Flow cytometry ; Fluid flow ; Fluids ; Good Manufacturing Practice ; Health services ; Hospital wastes ; Humans ; Immunomagnetic Separation - methods ; Joint surgery ; Medical personnel ; Medical research ; Medical wastes ; Medicine ; Mesenchymal stem cells ; Mesenchymal Stem Cells - cytology ; Mesenchymal Stem Cells - metabolism ; Mesenchyme ; Middle Aged ; Nerve Tissue Proteins - metabolism ; Osteogenesis ; Osteonectin ; Phenotype ; Plastics ; Polymerase chain reaction ; Product safety ; Quality ; R&D ; Receptors, Nerve Growth Factor - metabolism ; Regeneration ; Regeneration (physiology) ; Research & development ; Stem cells ; Stromal cells ; Studies ; Surgery ; Surgical equipment ; Technology ; Transplants & implants ; Waste materials ; Young Adult</subject><ispartof>PloS one, 2015-03, Vol.10 (3), p.e0117855-e0117855</ispartof><rights>2015 Cuthbert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Cuthbert et al 2015 Cuthbert et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c592t-9341d6e0d0fe8dbacec909874c1f5415cbf7a3459cc18ec6501583e9e5495b943</citedby><cites>FETCH-LOGICAL-c592t-9341d6e0d0fe8dbacec909874c1f5415cbf7a3459cc18ec6501583e9e5495b943</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356586/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356586/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25760857$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Menendez, Pablo</contributor><creatorcontrib>Cuthbert, Richard J</creatorcontrib><creatorcontrib>Giannoudis, Peter V</creatorcontrib><creatorcontrib>Wang, Xiao N</creatorcontrib><creatorcontrib>Nicholson, Lindsay</creatorcontrib><creatorcontrib>Pawson, David</creatorcontrib><creatorcontrib>Lubenko, Anatole</creatorcontrib><creatorcontrib>Tan, Hiang B</creatorcontrib><creatorcontrib>Dickinson, Anne</creatorcontrib><creatorcontrib>McGonagle, Dennis</creatorcontrib><creatorcontrib>Jones, Elena</creatorcontrib><title>Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells.
MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair.
Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs.
A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications.</description><subject>Adolescent</subject><subject>Adult</subject><subject>Aged</subject><subject>Arthritis</subject><subject>Autografts</subject><subject>Bone growth</subject><subject>Bone healing</subject><subject>Bone marrow</subject><subject>Bone morphogenetic protein 2</subject><subject>Bone Regeneration</subject><subject>CD105 antigen</subject><subject>CD45 antigen</subject><subject>CD73 antigen</subject><subject>CD90 antigen</subject><subject>Cell adhesion & migration</subject><subject>Cell culture</subject><subject>Cell Culture Techniques</subject><subject>Cell Proliferation</subject><subject>Child</subject><subject>Child, Preschool</subject><subject>Clinical trials</subject><subject>CXCL12 protein</subject><subject>Cytometry</subject><subject>Enrichment</subject><subject>Ethics</subject><subject>Feasibility studies</subject><subject>Femur</subject><subject>Femur Head - cytology</subject><subject>Flow cytometry</subject><subject>Fluid flow</subject><subject>Fluids</subject><subject>Good Manufacturing Practice</subject><subject>Health services</subject><subject>Hospital wastes</subject><subject>Humans</subject><subject>Immunomagnetic Separation - methods</subject><subject>Joint surgery</subject><subject>Medical personnel</subject><subject>Medical research</subject><subject>Medical wastes</subject><subject>Medicine</subject><subject>Mesenchymal stem cells</subject><subject>Mesenchymal Stem Cells - cytology</subject><subject>Mesenchymal Stem Cells - metabolism</subject><subject>Mesenchyme</subject><subject>Middle Aged</subject><subject>Nerve Tissue Proteins - metabolism</subject><subject>Osteogenesis</subject><subject>Osteonectin</subject><subject>Phenotype</subject><subject>Plastics</subject><subject>Polymerase chain reaction</subject><subject>Product safety</subject><subject>Quality</subject><subject>R&D</subject><subject>Receptors, Nerve Growth Factor - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Cuthbert, Richard J</au><au>Giannoudis, Peter V</au><au>Wang, Xiao N</au><au>Nicholson, Lindsay</au><au>Pawson, David</au><au>Lubenko, Anatole</au><au>Tan, Hiang B</au><au>Dickinson, Anne</au><au>McGonagle, Dennis</au><au>Jones, Elena</au><au>Menendez, Pablo</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-03-11</date><risdate>2015</risdate><volume>10</volume><issue>3</issue><spage>e0117855</spage><epage>e0117855</epage><pages>e0117855-e0117855</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells.
MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair.
Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs.
A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25760857</pmid><doi>10.1371/journal.pone.0117855</doi><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2015-03, Vol.10 (3), p.e0117855-e0117855 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1662426587 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Adolescent Adult Aged Arthritis Autografts Bone growth Bone healing Bone marrow Bone morphogenetic protein 2 Bone Regeneration CD105 antigen CD45 antigen CD73 antigen CD90 antigen Cell adhesion & migration Cell culture Cell Culture Techniques Cell Proliferation Child Child, Preschool Clinical trials CXCL12 protein Cytometry Enrichment Ethics Feasibility studies Femur Femur Head - cytology Flow cytometry Fluid flow Fluids Good Manufacturing Practice Health services Hospital wastes Humans Immunomagnetic Separation - methods Joint surgery Medical personnel Medical research Medical wastes Medicine Mesenchymal stem cells Mesenchymal Stem Cells - cytology Mesenchymal Stem Cells - metabolism Mesenchyme Middle Aged Nerve Tissue Proteins - metabolism Osteogenesis Osteonectin Phenotype Plastics Polymerase chain reaction Product safety Quality R&D Receptors, Nerve Growth Factor - metabolism Regeneration Regeneration (physiology) Research & development Stem cells Stromal cells Studies Surgery Surgical equipment Technology Transplants & implants Waste materials Young Adult |
| title | Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration |
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