A modified CD34+ hematopoietic stem and progenitor cell isolation strategy from cryopreserved human umbilical cord blood
| Autor: | Marina Tarunina, Frank Heuts, Peng Hua, Evangelia Rologi, Vickram Tittrea, Davide Grandolfo, Diana Hernandez, Marcia F Mata, Marc-Olivier Baradez, Robert Kallmeier, Swatisha Hirani, Yen Choo, Suzanne M. Watt, Enas Hassan |
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| Rok vydání: | 2019 |
| Předmět: |
Immunology
Population CD34 Antigens CD34 030204 cardiovascular system & hematology Biology Andrology 03 medical and health sciences Mice 0302 clinical medicine Immunology and Allergy Animals Humans Progenitor cell education Cryopreservation Gene Editing education.field_of_study Stem Cells Hematology Genetic Therapy Fetal Blood Hematopoietic Stem Cells Transplantation Haematopoiesis Cord blood Immunotherapy Stem cell Ex vivo 030215 immunology |
| Zdroj: | TransfusionREFERENCES. 59(12) |
| ISSN: | 1537-2995 |
| Popis: | Background Umbilical cord blood (UCB) is a source of hematopoietic stem cells for transplantation, offering an alternative for patients unable to find a matched adult donor. UCB is also a versatile source of hematopoietic stem and progenitor cells (hCD34 + HSPCs) for research into hematologic diseases, in vitro expansion, ex vivo gene therapy, and adoptive immunotherapy. For these studies, there is a need to isolate hCD34 + HSPCs from cryopreserved units, and protocols developed for isolation from fresh cord blood are unsuitable. Study design This study describes a modified method for isolating hCD34 + HSPCs from cryopreserved UCB. It uses the Plasmatherm system for thawing, followed by CD34 microbead magnetic-activated cell sorting isolation with a cell separation kit (Whole Blood Columns, Miltenyi Biotec). hCD34 + HSPC phenotypes and functionality were assessed in vitro and hematologic reconstitution determined in vivo in immunodeficient mice. Results Total nucleated cell recovery after thawing and washing was 44.7 ± 11.7%. Recovery of hCD34 + HSPCs after application of thawed cells to Whole Blood Columns was 77.5 ± 22.6%. When assessed in two independent laboratories, the hCD34+ cell purities were 71.7 ± 10.7% and 87.8 ± 2.4%. Transplantation of the enriched hCD34 + HSPCs into NSG mice revealed the presence of repopulating hematopoietic stem cells (estimated frequency of 0.07%) and multilineage engraftment. Conclusion This provides a simplified protocol for isolating high-purity human CD34 + HSPCs from banked UCB adaptable to current Good Manufacturing Practice. This protocol reduces the number of steps and associated risks and thus total production costs. Importantly, the isolated CD34 + HSPCs possess in vivo repopulating activity in immunodeficient mice, making them a suitable starting population for ex vivo culture and gene editing. |
| Databáze: | OpenAIRE |
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