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

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.

Clinical grade OK432-activated dendritic cells: in vitro characterization and tracking during intralymphatic delivery.

Dendritic cells (DC) are under intense preclinical and early clinical evaluation for the immunotherapy of cancer. However, the optimal culture conditions and route of delivery for DC vaccination have not been established. Here we describe the first human application of DC matured with the bacterial agent OK432 (OK-DC), using a short-term serum-free culture protocol, which generates mature DC from CD14+ precursors after 5 days. These cells were prepared within the framework of a National Blood Service facility, demonstrating that DC represent a product which is potentially deliverable alongside current standardized cell therapies within the UK National Health Service. In vitro analysis confirmed that OK-DC were mature, secreted tumor necrosis factor-alpha, interleukin-6, and interleukin-12, and stimulated both T cell and natural killer cell function. To explore effective delivery of OK-DC to lymph nodes, we performed an initial clinical tracking study of radioactively labeled, unpulsed OK-DC after intralymphatic injection into the dorsum of the foot. We showed that injected DC rapidly localized to ipsilateral pelvic lymph nodes, but did not disseminate to more distant nodes over a 48-hour period. There was no significant toxicity associated with OK-DC delivery. These results show that OK-DC are suitable for clinical use, and that intralymphatic delivery is feasible for localizing cells to sites where optimal priming of innate and adaptive antitumor immunity is likely to occur.

Single hospital experience of TRALI.

BACKGROUND: TRALI is a serious adverse effect of blood transfusion. There is evidence that the condition is underrecognized and underreported. STUDY DESIGN AND METHODS: This study was an observational study carried out in a single hospital. RESULTS: Eleven cases of TRALI were recognized over 12 years. In 10 cases the implicated donor unit was FFP and in 1 case uncertain. All implicated donors were parous women. In 4 cases the presumed causative antibodies were to an HLA class II antigen only. Specific anti-neutrophil antibodies, possibly causative, were detected in 1 case only. Ten of the 11 cases required mechanical ventilatory support. Five persons died as a result of the TRALI. The observed incidence of TRALI caused by FFP is 1 in 7900 units transfused. CONCLUSION: TRALI is the most common serious adverse effect of blood transfusion in our hospital. Antibodies to HLA class II antigens should be looked for routinely when investigating a possible case of TRALI.