Red blood cell depletion and enrichment of CD34+ hematopoietic progenitor cells from human umbilical cord blood using soybean agglutinin and CD34 immunoselection.
Realization of the full potential of human umbilical cord blood (HUCB) as a source of transplantable hematopoietic progenitor cells (HPC) will be contingent on the development of a reliable method for ex vivo cell processing and stem cell enrichment. A first step in stem cell enrichment protocols involves depletion of the red blood cells (RBC) in the sample. We have compared several RBC depletion methods on the basis of recovery of total nucleated cells. It was found that 3% gelatin was effective at depleting RBC with a nucleated cell recovery of 72.4 +/- 7.3% (mean +/- standard deviation [SD]) in the HUCB samples. Several lectins were also used for processing HUCB and compared for their ability to remove RBC. Of these, soybean agglutinin (SBA) was found to remove RBC without substantial loss of HPC. Moreover, sequential treatment of HUCB with 3% gelatin and the AIS MicroCELLector SBA resulted in a product with < 1% hematocrit, 57% reduction in T cells, 35% nucleated cell recovery, and relatively high yields of burst-forming units-erythroid (BFU-E) (61%) and colony-forming units-granulocyte/macrophage (CFU-GM) (58%) and -mixed (CFU-GEMM) (66%). In a separate series of studies, enrichment of the CD34+ subset in HUCB was accomplished by processing HUCB with Ficoll followed by sequential treatment with the AIS MicroCELLector SBA and AIS MicroCELLector CD34. The CD34+ fraction was enriched for BFU-E (14-fold), CFU-GM (nine-fold), and CFU-GEMM (five-fold) with an overall purity ¿ > or = 92% CD34+ by flow cytometry. This report demonstrates that 3% gelatin and the AIS MicroCELLector SBA can be combined as an ex vivo processing technique to reduce the volume of the HUCB product by depleting RBC and T cells while still maintaining a high recovery of HPC. Moreover, separation of highly enriched CD34+ cells from HUCB is achievable and opens up the possibility of using CD34+ cells from HUCB for ex vivo progenitor cell expansion for transplantation, transfusion, and gene therapy.
Nagler
A
A
Department of Bone Marrow Transplantation, Hadassah University Hospital, Jerusalem, Israel.
Peacock
M
M
Tantoco
M
M
Lamons
D
D
Okarma
T B
TB
Okrongly
D A
DA
D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003114:Colony-Forming Units Assay; D004912:Erythrocytes; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D005780:Gelatin; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D037102:Lectins / Q000378:metabolism; D037121:Plant Lectins; D011247:Pregnancy; D030262:Soybean Proteins; D010835:Phytohemagglutinins
D015703:Antigens, CD; D018952:Antigens, CD34; D005780:Gelatin; D037102:Lectins; D037121:Plant Lectins; D030262:Soybean Proteins; C028824:soybean lectin; D010835:Phytohemagglutinins