Cytotherapy, 2014; 16: 560e565
Recovery of CD45L/LinL/SSEA-4D very small embryonic-like stem cells by cord blood bank standard operating procedures
YU-JEN CHANG, KUEI-ERH TIEN, CHENG-HAO WEN, TZU-BOU HSIEH & SHIAW-MIN HWANG Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan Abstract Background aims. Very small embryonic-like (VSEL) stem cells are a rare cell population present in bone marrow, cord blood and other tissues that displays a distinct small cell size and the ability to give rise to cells of the three germ layers. VSEL stem cells were reported to be discarded in the red blood cell fraction by Ficoll-Paque density gradient centrifugation during the processing of bone marrow and cord blood specimens. However, most cord blood banks do not include density gradient centrifugation in their procedures while red blood cells are removed by Hespan sedimentation following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP). To clarify the retention of VSEL stem cells, we investigated the recovery of VSEL stem cells following COBLT SOP guidelines. Methods. The recovery of CD45/Lin/SSEA-4þ VSEL stem cells of umbilical cord blood was examined by flow cytometry before and after COBLT SOP processing, and relative expression of pluripotent genes was analyzed by quantitative polymerase chain reaction. Results. CD45/Lin/SSEA-4þ VSEL stem cells were mostly recovered in the final products following COBLT SOP guidelines. The expression of pluripotent genes could be maintained at >80% in products after hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) processing. Conclusions. The rare sub-population of CD45/Lin/SSEA-4þ VSEL stem cells survived after Hespan sedimentation. This finding suggests that umbilical cord blood units cryopreserved by COBLT SOP in cord blood banks should retain most VSEL stem cells present in the un-processed specimens. Key Words: cell recovery, cord blood bank standard operating procedures, Hespan sedimentation, very small embryonic stem cells
Introduction Umbilical cord blood (UCB), similar to bone marrow, is a rich source of hematopoietic stem cells for autologous and allogeneic transplantation. Besides hematopoietic lineage cells, studies have shown that many types of non-hematopoietic stem cells can be isolated from UCB and bone marrow, including mesenchymal (1,2), neural (3) and very small embryonic-like (VSEL) stem cells (4,5). VSEL stem cells were first identified in adult bone marrow (4) and subsequently were observed in other tissues and organs (6). VSEL stem cells are a rare cell subset with a small size (about 3e5 mm) and show a high nuclear-to-cytoplasmic ratio. In addition, VSEL stem cells express cell surface protein stagespecific embryonic antigen-4 (SSEA-4), a biomarker of human embryonic stem cells (7), and multiple nuclear pluripotent markers (Oct-4, Nanog and Sox2) (8,9). VSEL stem cells were also shown to give rise to cells of the three germ layers. They are
considered to be a unique stem cell population displaying pluripotency (4). Since the first successful therapeutic transplantation using cord blood in 1988 (10), both public and private cord blood banks have been set up worldwide for the cryopreservation of UCB at birth. It has been shown that when processing cord blood or bone marrow with the use of Ficoll-Paque, VSEL stem cells are discarded in the pelleted fraction (8). However, most cord blood banks process cord blood units following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP) standards (11). According to COBLT SOP, total nuclear cells are preserved while red blood cells (RBCs) are removed by hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) sedimentation, not density gradient centrifugation. In this study, we assessed the retention of VSEL stem cells in UCB following COBLT SOP guidelines. Our results demonstrated that most
Correspondence: Shiaw-Min Hwang, Ph.D., Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-pin Road, Hsinchu 30062, Taiwan. E-mail: hsm@firdi.org.tw (Received 9 May 2013; accepted 22 October 2013) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.10.009
Recovery of VSEL stem cells by COBLT SOP VSEL stem cells are retained in the final product of UCB. Methods Sample collection and processing UCB samples (n ¼ 6) were obtained from full-term deliveries. All donors were Taiwanese, and ages ranged from 25e35 years. Written informed consent was obtained from all donors, and all procedures were approved by the institutional review board of the Food Industry Research and Development Institute. UCB units were collected in standard 250-mL blood bags containing the anticoagulant, citrate phosphate, dextrose, and adenine. Each unit was weighed for total volume determination (range, 74e148 mL) and divided into two parts. The whole blood (WB) sample portion was obtained by withdrawal of each blood unit using a syringe, and the remaining portion was processed according to COBLT SOP. Briefly, RBCs were precipitated by adding Hespan, and the nonprecipitated cells were collected and centrifuged to obtain the leukocyte concentrate (LC). The LC was centrifuged and re-suspended in 20 mL phosphatebuffered saline (PBS) for further analysis. The nucleated cell numbers of each WB and LC specimen were determined using blood cell counter (KX-21; Sysmex, Kobe, Japan), and the total nucleated cell numbers in the un-processed and processed UCB units were calculated. Ficoll-Paque Plus density centrifugation To obtain mononuclear cells (MNCs), a 10-mL UCB sample was diluted 1:1 in PBS and top loaded on Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ, USA) (density, 1.077 g/mL) solution. After centrifugation at 2000 rpm for 40 min at room temperature, MNCs were collected from the interface. Flow cytometry and data analysis WB samples were incubated in RBC lysis buffer (BioLegend, San Diego, CA, USA) for 15 min at room temperature and washed twice in PBS. For determination of VSEL stem cells analysis by specific markers, 5e10 107 nucleated cells in the WB and LC samples were stained with anti-Lin cocktail 1 (CD3, CD14, CD16, CD19, CD20, CD56), fluorescein isothiocyanate, anti-CD45 allophycocyanin and anti-SSEA-4 phycoerythrin conjugated antibodies (BD Biosciences, San Diego, CA, USA) for 30 min at room temperature. Cells were fixed and permeabilized in 10% formaldehyde (Merck, Darmstadt, Germany) for 30 min. After washing by
561
PBS, 7-amino-actinomycin D (BD Biosciences) was added for 10 min to stain the cell nucleus. In this study, VSEL stem cells were immunophenotypically identified as the CD45/Lin/ SSEA-4þ cell subset. The basic principles of gating (Figure 1AeF) for VSEL stem cells were as follows: (i) A total events gate, P1, was created on the side scatter/ forward scatter dot plots that excluded most of the cellular debris; (ii) a P1-restricted P2 gate was generated on the side scatter/7-amino-actinomycin D dot plots that removed anucleated cells and the remaining cellular debris; (iii) a P2-restricted P3 gate on the side scatter/allophycocyanin dot plots was set to include only the CD45 cells; (iv) a P3-restricted P4 gate on the phycoerythrin/fluorescein isothiocyanate dot plots was set to include only the cells that expressed SSEA-4 but not Lin; (v) a P4-restricted P5 gate on the side scatter/forward scatter plots was set to include cell events sized
Recovery of CD45L/LinL/SSEA-4D very small embryonic-like stem cells by cord blood bank standard operating procedures
YU-JEN CHANG, KUEI-ERH TIEN, CHENG-HAO WEN, TZU-BOU HSIEH & SHIAW-MIN HWANG Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan Abstract Background aims. Very small embryonic-like (VSEL) stem cells are a rare cell population present in bone marrow, cord blood and other tissues that displays a distinct small cell size and the ability to give rise to cells of the three germ layers. VSEL stem cells were reported to be discarded in the red blood cell fraction by Ficoll-Paque density gradient centrifugation during the processing of bone marrow and cord blood specimens. However, most cord blood banks do not include density gradient centrifugation in their procedures while red blood cells are removed by Hespan sedimentation following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP). To clarify the retention of VSEL stem cells, we investigated the recovery of VSEL stem cells following COBLT SOP guidelines. Methods. The recovery of CD45/Lin/SSEA-4þ VSEL stem cells of umbilical cord blood was examined by flow cytometry before and after COBLT SOP processing, and relative expression of pluripotent genes was analyzed by quantitative polymerase chain reaction. Results. CD45/Lin/SSEA-4þ VSEL stem cells were mostly recovered in the final products following COBLT SOP guidelines. The expression of pluripotent genes could be maintained at >80% in products after hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) processing. Conclusions. The rare sub-population of CD45/Lin/SSEA-4þ VSEL stem cells survived after Hespan sedimentation. This finding suggests that umbilical cord blood units cryopreserved by COBLT SOP in cord blood banks should retain most VSEL stem cells present in the un-processed specimens. Key Words: cell recovery, cord blood bank standard operating procedures, Hespan sedimentation, very small embryonic stem cells
Introduction Umbilical cord blood (UCB), similar to bone marrow, is a rich source of hematopoietic stem cells for autologous and allogeneic transplantation. Besides hematopoietic lineage cells, studies have shown that many types of non-hematopoietic stem cells can be isolated from UCB and bone marrow, including mesenchymal (1,2), neural (3) and very small embryonic-like (VSEL) stem cells (4,5). VSEL stem cells were first identified in adult bone marrow (4) and subsequently were observed in other tissues and organs (6). VSEL stem cells are a rare cell subset with a small size (about 3e5 mm) and show a high nuclear-to-cytoplasmic ratio. In addition, VSEL stem cells express cell surface protein stagespecific embryonic antigen-4 (SSEA-4), a biomarker of human embryonic stem cells (7), and multiple nuclear pluripotent markers (Oct-4, Nanog and Sox2) (8,9). VSEL stem cells were also shown to give rise to cells of the three germ layers. They are
considered to be a unique stem cell population displaying pluripotency (4). Since the first successful therapeutic transplantation using cord blood in 1988 (10), both public and private cord blood banks have been set up worldwide for the cryopreservation of UCB at birth. It has been shown that when processing cord blood or bone marrow with the use of Ficoll-Paque, VSEL stem cells are discarded in the pelleted fraction (8). However, most cord blood banks process cord blood units following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP) standards (11). According to COBLT SOP, total nuclear cells are preserved while red blood cells (RBCs) are removed by hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) sedimentation, not density gradient centrifugation. In this study, we assessed the retention of VSEL stem cells in UCB following COBLT SOP guidelines. Our results demonstrated that most
Correspondence: Shiaw-Min Hwang, Ph.D., Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-pin Road, Hsinchu 30062, Taiwan. E-mail: hsm@firdi.org.tw (Received 9 May 2013; accepted 22 October 2013) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.10.009
Recovery of VSEL stem cells by COBLT SOP VSEL stem cells are retained in the final product of UCB. Methods Sample collection and processing UCB samples (n ¼ 6) were obtained from full-term deliveries. All donors were Taiwanese, and ages ranged from 25e35 years. Written informed consent was obtained from all donors, and all procedures were approved by the institutional review board of the Food Industry Research and Development Institute. UCB units were collected in standard 250-mL blood bags containing the anticoagulant, citrate phosphate, dextrose, and adenine. Each unit was weighed for total volume determination (range, 74e148 mL) and divided into two parts. The whole blood (WB) sample portion was obtained by withdrawal of each blood unit using a syringe, and the remaining portion was processed according to COBLT SOP. Briefly, RBCs were precipitated by adding Hespan, and the nonprecipitated cells were collected and centrifuged to obtain the leukocyte concentrate (LC). The LC was centrifuged and re-suspended in 20 mL phosphatebuffered saline (PBS) for further analysis. The nucleated cell numbers of each WB and LC specimen were determined using blood cell counter (KX-21; Sysmex, Kobe, Japan), and the total nucleated cell numbers in the un-processed and processed UCB units were calculated. Ficoll-Paque Plus density centrifugation To obtain mononuclear cells (MNCs), a 10-mL UCB sample was diluted 1:1 in PBS and top loaded on Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ, USA) (density, 1.077 g/mL) solution. After centrifugation at 2000 rpm for 40 min at room temperature, MNCs were collected from the interface. Flow cytometry and data analysis WB samples were incubated in RBC lysis buffer (BioLegend, San Diego, CA, USA) for 15 min at room temperature and washed twice in PBS. For determination of VSEL stem cells analysis by specific markers, 5e10 107 nucleated cells in the WB and LC samples were stained with anti-Lin cocktail 1 (CD3, CD14, CD16, CD19, CD20, CD56), fluorescein isothiocyanate, anti-CD45 allophycocyanin and anti-SSEA-4 phycoerythrin conjugated antibodies (BD Biosciences, San Diego, CA, USA) for 30 min at room temperature. Cells were fixed and permeabilized in 10% formaldehyde (Merck, Darmstadt, Germany) for 30 min. After washing by
561
PBS, 7-amino-actinomycin D (BD Biosciences) was added for 10 min to stain the cell nucleus. In this study, VSEL stem cells were immunophenotypically identified as the CD45/Lin/ SSEA-4þ cell subset. The basic principles of gating (Figure 1AeF) for VSEL stem cells were as follows: (i) A total events gate, P1, was created on the side scatter/ forward scatter dot plots that excluded most of the cellular debris; (ii) a P1-restricted P2 gate was generated on the side scatter/7-amino-actinomycin D dot plots that removed anucleated cells and the remaining cellular debris; (iii) a P2-restricted P3 gate on the side scatter/allophycocyanin dot plots was set to include only the CD45 cells; (iv) a P3-restricted P4 gate on the phycoerythrin/fluorescein isothiocyanate dot plots was set to include only the cells that expressed SSEA-4 but not Lin; (v) a P4-restricted P5 gate on the side scatter/forward scatter plots was set to include cell events sized
