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LSK derived LSK- cells have a high apoptotic rate related to survival regulation of hematopoietic and leukemic stem cells.

Peng C, Chen Y, Shan Y, Zhang H, Guo Z, Li D, Li S - PLoS ONE (2012)

Bottom Line: Here we show that the Lin(-)Sca-1(+)c-Kit(-) (LSK(-)) cell population derived from HSC-containing Lin(-)Sca-1(+)c-Kit(+) (LSK) cells has significantly higher numbers of apoptotic cells.In contrast, the LSK(-) population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK(-) population.These results indicate a potential function of the LSK(-) cells in the regulation of LSK cells and LSCs.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
A balanced pool of hematopoietic stem cells (HSCs) in bone marrow is tightly regulated, and this regulation is disturbed in hematopoietic malignancies such as chronic myeloid leukemia (CML). The underlying mechanisms are largely unknown. Here we show that the Lin(-)Sca-1(+)c-Kit(-) (LSK(-)) cell population derived from HSC-containing Lin(-)Sca-1(+)c-Kit(+) (LSK) cells has significantly higher numbers of apoptotic cells. Depletion of LSK cells by radiation or the cytotoxic chemical 5-fluorouracil results in an expansion of the LSK(-) population. In contrast, the LSK(-) population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK(-) population. The transition of LSK to LSK(-) cells is controlled by the Icsbp gene and its downstream gene Lyn, and regulation of this cellular transition is critical for the survival of normal LSK cells and LSCs. These results indicate a potential function of the LSK(-) cells in the regulation of LSK cells and LSCs.

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Related in: MedlinePlus

The LSK− cell population is derived from LSK cells and provides an apoptotic cellular pathway for LSK cells.(A) LSK (Lin−Sca1+c-Kit+) cells (1×103) and LSK− (Lin−Sca1+c-Kit−) cells (1×104) were sorted from bone marrow cells of CD45.1+ wild type (WT) mice by FACS, and transferred into lethal irradiated CD45.2+ WT recipient mice. 12 weeks later, bone marrow cells were collected and stained with antibodies for CD45.1, lineage markers, Sca−1 and c-Kit. Peripheral blood cells were also collected and stained with antibodies for CD45.1, Gr-1, Mac−1 and B220. (B) LSK cells were sorted from bone marrow of C56BL/6 (B6) mice by FACS and were irradiated (2000 cGy, once) or treated with 5-FU in vitro. The cells were cultured for 24 hours and then analyzed by FACS. (C) Apoptotic rates of progenitor and stem cells in vivo. Progenitor and stem cells were labeled with 7AAD and Annexin V, and analyzed by FACS (n = 5). **: p<0.01. (D) Cell cycle analysis of progenitor and stem cells in vivo. The cells were stained with Hoechst Blue, and the cells in the S+G2M phase were analyzed by FACS (n = 5). **: p<0.01.
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pone-0038614-g001: The LSK− cell population is derived from LSK cells and provides an apoptotic cellular pathway for LSK cells.(A) LSK (Lin−Sca1+c-Kit+) cells (1×103) and LSK− (Lin−Sca1+c-Kit−) cells (1×104) were sorted from bone marrow cells of CD45.1+ wild type (WT) mice by FACS, and transferred into lethal irradiated CD45.2+ WT recipient mice. 12 weeks later, bone marrow cells were collected and stained with antibodies for CD45.1, lineage markers, Sca−1 and c-Kit. Peripheral blood cells were also collected and stained with antibodies for CD45.1, Gr-1, Mac−1 and B220. (B) LSK cells were sorted from bone marrow of C56BL/6 (B6) mice by FACS and were irradiated (2000 cGy, once) or treated with 5-FU in vitro. The cells were cultured for 24 hours and then analyzed by FACS. (C) Apoptotic rates of progenitor and stem cells in vivo. Progenitor and stem cells were labeled with 7AAD and Annexin V, and analyzed by FACS (n = 5). **: p<0.01. (D) Cell cycle analysis of progenitor and stem cells in vivo. The cells were stained with Hoechst Blue, and the cells in the S+G2M phase were analyzed by FACS (n = 5). **: p<0.01.

Mentions: Because LSK− cells are derived from LSK cells and lack the ability to reconstitute lethally irradiated mice [5], we reasoned that this cell population is incapable of giving rise to LSK cells, progenitor cells and more mature cell lineages. To provide more supporting evidence, we transferred 1×104 LSK− cells or 1×103 LSK cells (as a positive control) from CD45.1 C57BL/6J (B6) mice into lethally irradiated CD45.2 B6 recipients. Three months after the bone marrow transplantation, FACS analysis showed that donor-derived CD45.1 LSK and LSK− cells were detected in bone marrow of recipients of CD45.1 LSK cells as expected, but neither cell population was detected in bone marrow of recipients of CD45.1 LSK− cells (Fig. 1A). In addition, no donor-derived mature myeloid (Gr1+CD45.1+ and Mac1+CD45.1+) and B-lymphoid cells (B220+CD45.1+) were detected in peripheral blood of recipients of CD45.1 LSK− cells (Fig. 1A). These results indicate that the LSK− cell population lacks long-term stem cell reconstitution function or an ability to give rise to LSK cells, consistent with the failure of LSK− cells to reconstitute lethally irradiated mice [5]. Although LSK cells give rise to LSK− cells, we wanted to know whether the LSK− cells could be directly differentiated from LSK or from other lineage negative populations. First, we sorted LSK cells from bone marrow of B6 mice, and treated the cells with the cytotoxic agent 5-FU or irradiation in vitro. The cells were then cultured for examining whether LSK− population could be produced directly from LSK cells. After the treatment with irradiation or 5-FU, we detected higher percentages of LSK− cells compared to the untreated control (Figure 1B). Second, we similarly treated Lin−Sca-1-c-Kit+ (LS−K; representing progenitor cells) with 5-FU or irradiation, and barely detected any LSK− cells (Fig. 1B), indicating that LS−K cells do not give rise to LSK− cells. Finally, we tested whether LSK− cell could arise from Lin−Sca-1-c-Kit− (LS−K−) cells by carrying out an in vivo reconstitution experiment. We transplanted 1×106 CD45.1 LS−K− cells into each CD45.2 recipient mouse, and continuously monitored CD45.1 donor cells in the recipient mice at 1, 2, 4, 8 and 16 weeks post bone marrow transplantation (BMT). At the first week, small percentages of CD45.1 cells were detected (Fig. S1A), but with time, CD45.1 cells disappeared in CD45.2 recipient mice (Fig. S1A, S1B), indicating that LS−K− cells do not give rise to any other populations including LSK− cells.


LSK derived LSK- cells have a high apoptotic rate related to survival regulation of hematopoietic and leukemic stem cells.

Peng C, Chen Y, Shan Y, Zhang H, Guo Z, Li D, Li S - PLoS ONE (2012)

The LSK− cell population is derived from LSK cells and provides an apoptotic cellular pathway for LSK cells.(A) LSK (Lin−Sca1+c-Kit+) cells (1×103) and LSK− (Lin−Sca1+c-Kit−) cells (1×104) were sorted from bone marrow cells of CD45.1+ wild type (WT) mice by FACS, and transferred into lethal irradiated CD45.2+ WT recipient mice. 12 weeks later, bone marrow cells were collected and stained with antibodies for CD45.1, lineage markers, Sca−1 and c-Kit. Peripheral blood cells were also collected and stained with antibodies for CD45.1, Gr-1, Mac−1 and B220. (B) LSK cells were sorted from bone marrow of C56BL/6 (B6) mice by FACS and were irradiated (2000 cGy, once) or treated with 5-FU in vitro. The cells were cultured for 24 hours and then analyzed by FACS. (C) Apoptotic rates of progenitor and stem cells in vivo. Progenitor and stem cells were labeled with 7AAD and Annexin V, and analyzed by FACS (n = 5). **: p<0.01. (D) Cell cycle analysis of progenitor and stem cells in vivo. The cells were stained with Hoechst Blue, and the cells in the S+G2M phase were analyzed by FACS (n = 5). **: p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366951&req=5

pone-0038614-g001: The LSK− cell population is derived from LSK cells and provides an apoptotic cellular pathway for LSK cells.(A) LSK (Lin−Sca1+c-Kit+) cells (1×103) and LSK− (Lin−Sca1+c-Kit−) cells (1×104) were sorted from bone marrow cells of CD45.1+ wild type (WT) mice by FACS, and transferred into lethal irradiated CD45.2+ WT recipient mice. 12 weeks later, bone marrow cells were collected and stained with antibodies for CD45.1, lineage markers, Sca−1 and c-Kit. Peripheral blood cells were also collected and stained with antibodies for CD45.1, Gr-1, Mac−1 and B220. (B) LSK cells were sorted from bone marrow of C56BL/6 (B6) mice by FACS and were irradiated (2000 cGy, once) or treated with 5-FU in vitro. The cells were cultured for 24 hours and then analyzed by FACS. (C) Apoptotic rates of progenitor and stem cells in vivo. Progenitor and stem cells were labeled with 7AAD and Annexin V, and analyzed by FACS (n = 5). **: p<0.01. (D) Cell cycle analysis of progenitor and stem cells in vivo. The cells were stained with Hoechst Blue, and the cells in the S+G2M phase were analyzed by FACS (n = 5). **: p<0.01.
Mentions: Because LSK− cells are derived from LSK cells and lack the ability to reconstitute lethally irradiated mice [5], we reasoned that this cell population is incapable of giving rise to LSK cells, progenitor cells and more mature cell lineages. To provide more supporting evidence, we transferred 1×104 LSK− cells or 1×103 LSK cells (as a positive control) from CD45.1 C57BL/6J (B6) mice into lethally irradiated CD45.2 B6 recipients. Three months after the bone marrow transplantation, FACS analysis showed that donor-derived CD45.1 LSK and LSK− cells were detected in bone marrow of recipients of CD45.1 LSK cells as expected, but neither cell population was detected in bone marrow of recipients of CD45.1 LSK− cells (Fig. 1A). In addition, no donor-derived mature myeloid (Gr1+CD45.1+ and Mac1+CD45.1+) and B-lymphoid cells (B220+CD45.1+) were detected in peripheral blood of recipients of CD45.1 LSK− cells (Fig. 1A). These results indicate that the LSK− cell population lacks long-term stem cell reconstitution function or an ability to give rise to LSK cells, consistent with the failure of LSK− cells to reconstitute lethally irradiated mice [5]. Although LSK cells give rise to LSK− cells, we wanted to know whether the LSK− cells could be directly differentiated from LSK or from other lineage negative populations. First, we sorted LSK cells from bone marrow of B6 mice, and treated the cells with the cytotoxic agent 5-FU or irradiation in vitro. The cells were then cultured for examining whether LSK− population could be produced directly from LSK cells. After the treatment with irradiation or 5-FU, we detected higher percentages of LSK− cells compared to the untreated control (Figure 1B). Second, we similarly treated Lin−Sca-1-c-Kit+ (LS−K; representing progenitor cells) with 5-FU or irradiation, and barely detected any LSK− cells (Fig. 1B), indicating that LS−K cells do not give rise to LSK− cells. Finally, we tested whether LSK− cell could arise from Lin−Sca-1-c-Kit− (LS−K−) cells by carrying out an in vivo reconstitution experiment. We transplanted 1×106 CD45.1 LS−K− cells into each CD45.2 recipient mouse, and continuously monitored CD45.1 donor cells in the recipient mice at 1, 2, 4, 8 and 16 weeks post bone marrow transplantation (BMT). At the first week, small percentages of CD45.1 cells were detected (Fig. S1A), but with time, CD45.1 cells disappeared in CD45.2 recipient mice (Fig. S1A, S1B), indicating that LS−K− cells do not give rise to any other populations including LSK− cells.

Bottom Line: Here we show that the Lin(-)Sca-1(+)c-Kit(-) (LSK(-)) cell population derived from HSC-containing Lin(-)Sca-1(+)c-Kit(+) (LSK) cells has significantly higher numbers of apoptotic cells.In contrast, the LSK(-) population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK(-) population.These results indicate a potential function of the LSK(-) cells in the regulation of LSK cells and LSCs.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
A balanced pool of hematopoietic stem cells (HSCs) in bone marrow is tightly regulated, and this regulation is disturbed in hematopoietic malignancies such as chronic myeloid leukemia (CML). The underlying mechanisms are largely unknown. Here we show that the Lin(-)Sca-1(+)c-Kit(-) (LSK(-)) cell population derived from HSC-containing Lin(-)Sca-1(+)c-Kit(+) (LSK) cells has significantly higher numbers of apoptotic cells. Depletion of LSK cells by radiation or the cytotoxic chemical 5-fluorouracil results in an expansion of the LSK(-) population. In contrast, the LSK(-) population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK(-) population. The transition of LSK to LSK(-) cells is controlled by the Icsbp gene and its downstream gene Lyn, and regulation of this cellular transition is critical for the survival of normal LSK cells and LSCs. These results indicate a potential function of the LSK(-) cells in the regulation of LSK cells and LSCs.

Show MeSH
Related in: MedlinePlus