Delineating the effects of 5-fluorouracil and follicle-stimulating hormone on mouse bone marrow stem/progenitor cells.
BACKGROUND
Pluripotent, Lin(-)/CD45(-)/Sca-1(+) very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin(-)/CD45(+)/Sca-1(+) hematopoietic stem cells (HSCs) get eliminated. In the present study, we provide further evidence for the existence of VSELs in mouse BM and have also examined the effects of a chemotherapeutic agent (5-fluorouracil (5-FU)) and gonadotropin hormone (follicle-stimulating hormone (FSH)) on BM stem/progenitor cells.


METHODS
VSELs and HSCs were characterized in intact BM. Swiss mice were injected with 5-FU (150 mg/kg) and sacrificed on 2, 4, and 10 days (D2, D4, and D10) post treatment to examine changes in BM histology and effects on VSELs and HSCs by a multiparametric approach. The effect of FSH (5 IU) administered 48 h after 5-FU treatment was also studied. Bromodeoxyuridine (BrdU) incorporation, cell cycle analysis, and colony-forming unit (CFU) assay were carried out to understand the functional potential of stem/progenitor cells towards regeneration of chemoablated marrow.


RESULTS
Nuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN(-)/CD45(-) VSELs and slightly larger LIN(-)/CD45(+) HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10. Nuclear OCT-4 and PCNA-positive VSELs were detected in chemoablated bone sections near the endosteal region. VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna). Cells that survived 5-FU formed colonies in vitro. Both VSELs and HSCs expressed FSH receptors and FSH treatment enhanced hematopoietic recovery by 72 h.


CONCLUSIONS
Both VSELs and HSCs were activated in response to the stress created by 5-FU and FSH enhanced hematopoietic recovery by at least 72 h in 5-FU-treated mice. VSELs are the most primitive pluripotent stem cells in BM that self-renew and give rise to HSCs under stress, and HSCs further divide rapidly and differentiate to maintain homeostasis. The study provides a novel insight into basic hematopoiesis and has clinical relevance.
Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai, 400 012, India.
Ambreen
A
Shaikh
Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai, 400 012, India. deepa.bhartiya@yahoo.in.
Deepa
D
Bhartiya
Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai, 400 012, India.
Sona
S
Kapoor
Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai, 400 012, India.
Harshada
H
Nimkar
D000818:Animals; D000067696:Ataxin-1 / Q000235:genetics / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D048429:Cell Size; D002868:Chromosomal Proteins, Non-Histone; D005472:Fluorouracil / Q000494:pharmacology*; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D015870:Gene Expression; D006410:Hematopoiesis / Q000187:drug effects* / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016130:Immunophenotyping; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism; D016256:Lewis X Antigen / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011962:Receptors, FSH / Q000235:genetics / Q000378:metabolism
D000067696:Ataxin-1; C000593750:Atxn1 protein, mouse; D002868:Chromosomal Proteins, Non-Histone; C466105:DPPA3 protein, human; D016256:Lewis X Antigen; D008565:Membrane Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D011962:Receptors, FSH; C462181:fragilis protein, mouse; D005640:Follicle Stimulating Hormone; D017493:Leukocyte Common Antigens; C516770:Ptprc protein, mouse; D005472:Fluorouracil
5-Fluorouracil; Bone marrow; Follicle-stimulating hormone; Hematopoietic stem cells; OCT-4; Very small embryonic-like stem cells