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Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration.

Cuthbert RJ, Giannoudis PV, Wang XN, Nicholson L, Pawson D, Lubenko A, Tan HB, Dickinson A, McGonagle D, Jones E - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.

ABSTRACT

Introduction: 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.

Materials and methods: 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.

Results: 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.

Discussion: 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.

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Functional analysis, phenotypic profile and transcripts expression of CD271 selected MSCs from BM, FH and RIA.Differentiation potential of PA-MSCs and CD271-MSCs. Osteogenesis, adipogenesis and chondrogenesis was measured on day-21 post-induction by staining with alkaline phosphatase/von Kossa, Oil Red O and Alcian Blue, respectively in cells selected from BM (A), FH (B) and RIA (C), all size bars represent 500μm. Phenotypic analysis of the CD45−/lowCD271+ population observed in BM (D), FH (E) and RIA (F). Analysis of the total expression of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts relative to HPRT in BM (G), FH (H) and RIA (I) pre (black bars) and post (grey bars) CD271 enrichment. (All data n = 3).
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pone.0117855.g004: Functional analysis, phenotypic profile and transcripts expression of CD271 selected MSCs from BM, FH and RIA.Differentiation potential of PA-MSCs and CD271-MSCs. Osteogenesis, adipogenesis and chondrogenesis was measured on day-21 post-induction by staining with alkaline phosphatase/von Kossa, Oil Red O and Alcian Blue, respectively in cells selected from BM (A), FH (B) and RIA (C), all size bars represent 500μm. Phenotypic analysis of the CD45−/lowCD271+ population observed in BM (D), FH (E) and RIA (F). Analysis of the total expression of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts relative to HPRT in BM (G), FH (H) and RIA (I) pre (black bars) and post (grey bars) CD271 enrichment. (All data n = 3).

Mentions: The functional and phenotypic characteristics of CD271 selected cells from BM, FH and RIA were compared next. In every case CD271 selection resulted in the isolation of cells capable of osteogenic, adipogenic and chondrogenic differentiation (Fig. 4A-C). Differentiation capacity was comparable to MSCs selected by plastic adherence as assessed by positive staining using alkaline phosphatase/von Kossa, Oil Red O and Alcian Blue respectively.


Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration.

Cuthbert RJ, Giannoudis PV, Wang XN, Nicholson L, Pawson D, Lubenko A, Tan HB, Dickinson A, McGonagle D, Jones E - PLoS ONE (2015)

Functional analysis, phenotypic profile and transcripts expression of CD271 selected MSCs from BM, FH and RIA.Differentiation potential of PA-MSCs and CD271-MSCs. Osteogenesis, adipogenesis and chondrogenesis was measured on day-21 post-induction by staining with alkaline phosphatase/von Kossa, Oil Red O and Alcian Blue, respectively in cells selected from BM (A), FH (B) and RIA (C), all size bars represent 500μm. Phenotypic analysis of the CD45−/lowCD271+ population observed in BM (D), FH (E) and RIA (F). Analysis of the total expression of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts relative to HPRT in BM (G), FH (H) and RIA (I) pre (black bars) and post (grey bars) CD271 enrichment. (All data n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356586&req=5

pone.0117855.g004: Functional analysis, phenotypic profile and transcripts expression of CD271 selected MSCs from BM, FH and RIA.Differentiation potential of PA-MSCs and CD271-MSCs. Osteogenesis, adipogenesis and chondrogenesis was measured on day-21 post-induction by staining with alkaline phosphatase/von Kossa, Oil Red O and Alcian Blue, respectively in cells selected from BM (A), FH (B) and RIA (C), all size bars represent 500μm. Phenotypic analysis of the CD45−/lowCD271+ population observed in BM (D), FH (E) and RIA (F). Analysis of the total expression of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts relative to HPRT in BM (G), FH (H) and RIA (I) pre (black bars) and post (grey bars) CD271 enrichment. (All data n = 3).
Mentions: The functional and phenotypic characteristics of CD271 selected cells from BM, FH and RIA were compared next. In every case CD271 selection resulted in the isolation of cells capable of osteogenic, adipogenic and chondrogenic differentiation (Fig. 4A-C). Differentiation capacity was comparable to MSCs selected by plastic adherence as assessed by positive staining using alkaline phosphatase/von Kossa, Oil Red O and Alcian Blue respectively.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.

ABSTRACT

Introduction: 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.

Materials and methods: 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.

Results: 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.

Discussion: 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.

Show MeSH
Related in: MedlinePlus