Culture conditions that stimulate expansion of normal hematopoietic progenitors from chronic myelogenous leukemia marrow

R. M. Lemoli, M. Fortuna, M. Amabile, A. Fortuna, D. Rondelli, G. Martinelli, S. Tura

Research output: Contribution to journalArticle

Abstract

In this study, CD34+ cells and CD34+ I)R- tells were sorted from the bone marrow (BM) of chronic phase CML patients at diagnosis. Primitive hematopoietic celK were tested for their colony fanning ability 111 response to early and iniermeduite-Uite colony stimulating factors (CSFs), and the presence of the BCR-ABL transcript in individually plucked colonies was detected by nested Reverse Traiwcriptase-Polj niera se Chain Reaction (RT-PCR). B-actm and ABL transcripts were used as internal controls Molecular analysis revealed that 673 ±16% SD of CD34+ colonies and 55.6 ±9% SI) of CD34+DR- colonies stimulated by phytohemagglntinin lymphocyte-conditioned medium (PHA-LCM), in which actin and ABL mRNA were detectable, did not express the product of the BCR-ABL gene. The absolute number and the clonogenic efficiency of CML DRcells was significantly' lower than that of comparable DR- cells from normal donors However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, fl.-l 1. IL-3, used as single factors in presence of erythropoietm (EPO) SCF and EPO generated 134 ±18% of colon) growth induced by PHA-LCM. This result was almost entirely due to erythroid progenitors SCI and EPO produced nearly Mold increase of burst-fanning unit-erythroid (BFU-I-) compared to PHA-LCM. Conversely, optimal stimulation ot CD34+ cells and CO34+DKcells from normal donors required co-incubation with 3 or more CSFs. Stroma-noncontact long-term cultures were then established in presence of exogenous CSFs and human irradiated allogeneic stromal layers or the engineered niurine stromal cell line M2-10B4 to produce G-CSF and 1I.-3 In this system, the combination of SCF and IL-3 induced a 25.4 ±5SD- , 40 ±6SD- and 20 5 ±6SD-tbld increase of colony-forming unit cells |CI-'U-C), at week 2, 4 and 5, respectively. At the same time-points, the number o] primitive long-term culture initiating cells {LTC-ICj showed a 4 ±2 SD-. 3.3 ±1 5SDand 2.3 it ISD-fold increase as compared to baseline value. The addition of 11,-11 to SCF/IL-3 combination neither further increased the expansion of CFU-C nor the survi\al of LTC-IC. Analysis of the BCR-ABL transcripts at the level of single (.dome demonstrated that 63 ±9% SD and 93 ±3% SD CFü-C at week 4 and 5, respectively, did not express the fusion gene whereas leukemic LTC'-1C disappeared from the culture b\ \veek 2 In summary, our results suggest that leukemic CD34+DR-lin- cells behave as more mature normal progenitors with respect to sensitivity to CSFs and proliterative potential, hi addition, we established culture conditions which allow the sclccinc expansion of benign hematopoieiic cells coexisting with leukemic progenitors.

Original languageEnglish
Pages (from-to)809
Number of pages1
JournalExperimental Hematology
Volume25
Issue number8
Publication statusPublished - 1997

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Colony-Stimulating Factors
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Bone Marrow
Interleukin-3
Conditioned Culture Medium
Lymphocytes
Tissue Donors
Gene Fusion
Stromal Cells
Actins
Colon
Fungi
Stem Cells
Cell Culture Techniques
Cell Proliferation
Cell Line
Messenger RNA
Growth
Genes

ASJC Scopus subject areas

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

Cite this

Lemoli, R. M., Fortuna, M., Amabile, M., Fortuna, A., Rondelli, D., Martinelli, G., & Tura, S. (1997). Culture conditions that stimulate expansion of normal hematopoietic progenitors from chronic myelogenous leukemia marrow. Experimental Hematology, 25(8), 809.

Culture conditions that stimulate expansion of normal hematopoietic progenitors from chronic myelogenous leukemia marrow. / Lemoli, R. M.; Fortuna, M.; Amabile, M.; Fortuna, A.; Rondelli, D.; Martinelli, G.; Tura, S.

In: Experimental Hematology, Vol. 25, No. 8, 1997, p. 809.

Research output: Contribution to journalArticle

Lemoli, RM, Fortuna, M, Amabile, M, Fortuna, A, Rondelli, D, Martinelli, G & Tura, S 1997, 'Culture conditions that stimulate expansion of normal hematopoietic progenitors from chronic myelogenous leukemia marrow', Experimental Hematology, vol. 25, no. 8, pp. 809.
Lemoli, R. M. ; Fortuna, M. ; Amabile, M. ; Fortuna, A. ; Rondelli, D. ; Martinelli, G. ; Tura, S. / Culture conditions that stimulate expansion of normal hematopoietic progenitors from chronic myelogenous leukemia marrow. In: Experimental Hematology. 1997 ; Vol. 25, No. 8. pp. 809.
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T1 - Culture conditions that stimulate expansion of normal hematopoietic progenitors from chronic myelogenous leukemia marrow

AU - Lemoli, R. M.

AU - Fortuna, M.

AU - Amabile, M.

AU - Fortuna, A.

AU - Rondelli, D.

AU - Martinelli, G.

AU - Tura, S.

PY - 1997

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N2 - In this study, CD34+ cells and CD34+ I)R- tells were sorted from the bone marrow (BM) of chronic phase CML patients at diagnosis. Primitive hematopoietic celK were tested for their colony fanning ability 111 response to early and iniermeduite-Uite colony stimulating factors (CSFs), and the presence of the BCR-ABL transcript in individually plucked colonies was detected by nested Reverse Traiwcriptase-Polj niera se Chain Reaction (RT-PCR). B-actm and ABL transcripts were used as internal controls Molecular analysis revealed that 673 ±16% SD of CD34+ colonies and 55.6 ±9% SI) of CD34+DR- colonies stimulated by phytohemagglntinin lymphocyte-conditioned medium (PHA-LCM), in which actin and ABL mRNA were detectable, did not express the product of the BCR-ABL gene. The absolute number and the clonogenic efficiency of CML DRcells was significantly' lower than that of comparable DR- cells from normal donors However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, fl.-l 1. IL-3, used as single factors in presence of erythropoietm (EPO) SCF and EPO generated 134 ±18% of colon) growth induced by PHA-LCM. This result was almost entirely due to erythroid progenitors SCI and EPO produced nearly Mold increase of burst-fanning unit-erythroid (BFU-I-) compared to PHA-LCM. Conversely, optimal stimulation ot CD34+ cells and CO34+DKcells from normal donors required co-incubation with 3 or more CSFs. Stroma-noncontact long-term cultures were then established in presence of exogenous CSFs and human irradiated allogeneic stromal layers or the engineered niurine stromal cell line M2-10B4 to produce G-CSF and 1I.-3 In this system, the combination of SCF and IL-3 induced a 25.4 ±5SD- , 40 ±6SD- and 20 5 ±6SD-tbld increase of colony-forming unit cells |CI-'U-C), at week 2, 4 and 5, respectively. At the same time-points, the number o] primitive long-term culture initiating cells {LTC-ICj showed a 4 ±2 SD-. 3.3 ±1 5SDand 2.3 it ISD-fold increase as compared to baseline value. The addition of 11,-11 to SCF/IL-3 combination neither further increased the expansion of CFU-C nor the survi\al of LTC-IC. Analysis of the BCR-ABL transcripts at the level of single (.dome demonstrated that 63 ±9% SD and 93 ±3% SD CFü-C at week 4 and 5, respectively, did not express the fusion gene whereas leukemic LTC'-1C disappeared from the culture b\ \veek 2 In summary, our results suggest that leukemic CD34+DR-lin- cells behave as more mature normal progenitors with respect to sensitivity to CSFs and proliterative potential, hi addition, we established culture conditions which allow the sclccinc expansion of benign hematopoieiic cells coexisting with leukemic progenitors.

AB - In this study, CD34+ cells and CD34+ I)R- tells were sorted from the bone marrow (BM) of chronic phase CML patients at diagnosis. Primitive hematopoietic celK were tested for their colony fanning ability 111 response to early and iniermeduite-Uite colony stimulating factors (CSFs), and the presence of the BCR-ABL transcript in individually plucked colonies was detected by nested Reverse Traiwcriptase-Polj niera se Chain Reaction (RT-PCR). B-actm and ABL transcripts were used as internal controls Molecular analysis revealed that 673 ±16% SD of CD34+ colonies and 55.6 ±9% SI) of CD34+DR- colonies stimulated by phytohemagglntinin lymphocyte-conditioned medium (PHA-LCM), in which actin and ABL mRNA were detectable, did not express the product of the BCR-ABL gene. The absolute number and the clonogenic efficiency of CML DRcells was significantly' lower than that of comparable DR- cells from normal donors However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, fl.-l 1. IL-3, used as single factors in presence of erythropoietm (EPO) SCF and EPO generated 134 ±18% of colon) growth induced by PHA-LCM. This result was almost entirely due to erythroid progenitors SCI and EPO produced nearly Mold increase of burst-fanning unit-erythroid (BFU-I-) compared to PHA-LCM. Conversely, optimal stimulation ot CD34+ cells and CO34+DKcells from normal donors required co-incubation with 3 or more CSFs. Stroma-noncontact long-term cultures were then established in presence of exogenous CSFs and human irradiated allogeneic stromal layers or the engineered niurine stromal cell line M2-10B4 to produce G-CSF and 1I.-3 In this system, the combination of SCF and IL-3 induced a 25.4 ±5SD- , 40 ±6SD- and 20 5 ±6SD-tbld increase of colony-forming unit cells |CI-'U-C), at week 2, 4 and 5, respectively. At the same time-points, the number o] primitive long-term culture initiating cells {LTC-ICj showed a 4 ±2 SD-. 3.3 ±1 5SDand 2.3 it ISD-fold increase as compared to baseline value. The addition of 11,-11 to SCF/IL-3 combination neither further increased the expansion of CFU-C nor the survi\al of LTC-IC. Analysis of the BCR-ABL transcripts at the level of single (.dome demonstrated that 63 ±9% SD and 93 ±3% SD CFü-C at week 4 and 5, respectively, did not express the fusion gene whereas leukemic LTC'-1C disappeared from the culture b\ \veek 2 In summary, our results suggest that leukemic CD34+DR-lin- cells behave as more mature normal progenitors with respect to sensitivity to CSFs and proliterative potential, hi addition, we established culture conditions which allow the sclccinc expansion of benign hematopoieiic cells coexisting with leukemic progenitors.

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