Modified in vitro conditions for cord blood-derived long-term culture-initiating cells

Marina Podestà, Giovanna Piaggio, Anna Pitto, Elena Zocchi, Monica Soracco, Francesco Frassoni, Silvia Luchetti, Enrica Painelli, Andrea Bacigalupo

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Objective. The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. Materials and Methods. We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37°C and 33°C) and supportive stromal cell lines (NIH3T3 and M210-B4). Results. The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p <10-4 and p <0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 ± 5.6/104 MNC) compared to bone marrow (7 ± 3.8/104 MNC; p <0.005) and peripheral blood (15.2 ± 11.1/104 MNC; p <10-4). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 ± 3.8/106 MNC) in cord blood with respect to bone marrow (40.7 ± 7.4/106 MNC; p <10-4) and peripheral blood (28.8 ± 3.8/106 MNC; p <0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 ± 7.7 colonies vs 5.9 ± 5 vs 3.2 ± 2.2 colonies; p <0.02 and p <0.001 respectively).Conclusions. Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.

Original languageEnglish
Pages (from-to)309-314
Number of pages6
JournalExperimental Hematology
Volume29
Issue number3
DOIs
Publication statusPublished - 2001

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Fetal Blood
Cell Culture Techniques
Bone Marrow
Granulocytes
Erythroid Precursor Cells
Megakaryocytes
Granulocyte Colony-Stimulating Factor
Macrophages
In Vitro Techniques
Colony-Forming Units Assay
Granulocyte-Macrophage Progenitor Cells
Temperature
Stromal Cells
Growth
Transplantation
Cell Line

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

Modified in vitro conditions for cord blood-derived long-term culture-initiating cells. / Podestà, Marina; Piaggio, Giovanna; Pitto, Anna; Zocchi, Elena; Soracco, Monica; Frassoni, Francesco; Luchetti, Silvia; Painelli, Enrica; Bacigalupo, Andrea.

In: Experimental Hematology, Vol. 29, No. 3, 2001, p. 309-314.

Research output: Contribution to journalArticle

Podestà, M, Piaggio, G, Pitto, A, Zocchi, E, Soracco, M, Frassoni, F, Luchetti, S, Painelli, E & Bacigalupo, A 2001, 'Modified in vitro conditions for cord blood-derived long-term culture-initiating cells', Experimental Hematology, vol. 29, no. 3, pp. 309-314. https://doi.org/10.1016/S0301-472X(00)00678-0
Podestà, Marina ; Piaggio, Giovanna ; Pitto, Anna ; Zocchi, Elena ; Soracco, Monica ; Frassoni, Francesco ; Luchetti, Silvia ; Painelli, Enrica ; Bacigalupo, Andrea. / Modified in vitro conditions for cord blood-derived long-term culture-initiating cells. In: Experimental Hematology. 2001 ; Vol. 29, No. 3. pp. 309-314.
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abstract = "Objective. The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. Materials and Methods. We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37°C and 33°C) and supportive stromal cell lines (NIH3T3 and M210-B4). Results. The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p <10-4 and p <0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 ± 5.6/104 MNC) compared to bone marrow (7 ± 3.8/104 MNC; p <0.005) and peripheral blood (15.2 ± 11.1/104 MNC; p <10-4). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 ± 3.8/106 MNC) in cord blood with respect to bone marrow (40.7 ± 7.4/106 MNC; p <10-4) and peripheral blood (28.8 ± 3.8/106 MNC; p <0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 ± 7.7 colonies vs 5.9 ± 5 vs 3.2 ± 2.2 colonies; p <0.02 and p <0.001 respectively).Conclusions. Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.",
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T1 - Modified in vitro conditions for cord blood-derived long-term culture-initiating cells

AU - Podestà, Marina

AU - Piaggio, Giovanna

AU - Pitto, Anna

AU - Zocchi, Elena

AU - Soracco, Monica

AU - Frassoni, Francesco

AU - Luchetti, Silvia

AU - Painelli, Enrica

AU - Bacigalupo, Andrea

PY - 2001

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N2 - Objective. The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. Materials and Methods. We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37°C and 33°C) and supportive stromal cell lines (NIH3T3 and M210-B4). Results. The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p <10-4 and p <0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 ± 5.6/104 MNC) compared to bone marrow (7 ± 3.8/104 MNC; p <0.005) and peripheral blood (15.2 ± 11.1/104 MNC; p <10-4). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 ± 3.8/106 MNC) in cord blood with respect to bone marrow (40.7 ± 7.4/106 MNC; p <10-4) and peripheral blood (28.8 ± 3.8/106 MNC; p <0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 ± 7.7 colonies vs 5.9 ± 5 vs 3.2 ± 2.2 colonies; p <0.02 and p <0.001 respectively).Conclusions. Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.

AB - Objective. The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. Materials and Methods. We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37°C and 33°C) and supportive stromal cell lines (NIH3T3 and M210-B4). Results. The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p <10-4 and p <0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 ± 5.6/104 MNC) compared to bone marrow (7 ± 3.8/104 MNC; p <0.005) and peripheral blood (15.2 ± 11.1/104 MNC; p <10-4). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 ± 3.8/106 MNC) in cord blood with respect to bone marrow (40.7 ± 7.4/106 MNC; p <10-4) and peripheral blood (28.8 ± 3.8/106 MNC; p <0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 ± 7.7 colonies vs 5.9 ± 5 vs 3.2 ± 2.2 colonies; p <0.02 and p <0.001 respectively).Conclusions. Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.

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