PMID- 8035601
OWN - NLM
STAT- MEDLINE
DCOM- 19940812
LR  - 20131121
IS  - 0887-6924 (Print)
IS  - 0887-6924 (Linking)
VI  - 8
IP  - 7
DP  - 1994 Jul
TI  - Frequency analysis of human primitive haematopoietic stem cell subsets using a 
      cobblestone area forming cell assay.
PG  - 1095-104
AB  - Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of 
      primitive haematopoietic stem cells (HSC) for long-term production of clonable 
      progenitors. We have developed a limiting dilution type LTBMC assay allowing 
      frequency analysis of transiently repopulating HSC and long-term culture 
      initiating cells (LTC-IC) without the necessity to replate large numbers of 
      wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC), or BMCs sorted on 
      CD34 or HLA-DR expression, or Rh123 retention, (input range 40-70,000 
      CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated 
      adherent layers formed by a novel murine preadipose cell line (FBMD-1). The 
      percentage of wells with at least one phase-dark haematopoietic clone 
      (cobblestone area, CA) beneath the stromal layer was weekly determined for at 
      least 8 weeks, and CA-forming cell (CAFC) frequencies were calculated using 
      Poisson statistics. Parallel LTBMCs of the same samples were weekly assessed for 
      supernate CFU-GM/BFU-E production. Weekly addition of rhIL-3 with rhG-CSF 
      supported a high average clonogenic output per CA and dramatically increased CA 
      size, but did not significantly alter the apparent CAFC frequency. The generation 
      of CFU-GM per CA was constant over a period of 6 weeks with weekly means of eight 
      normal BM samples, ranging between 5-16. At week 6 the mean CAFC frequency was 29 
      (1 SEM, 8.8)/10(5). Early appearing CAFC were highly sensitive to 5-FU, and were 
      contained over the full Rh123 and HLA-DR fluorescence profile of CD34pos cells, 
      whereas CAFC week 5-8 were predominantly contained in the CD34pos Rh123dull 
      HLA-DRlow fraction in agreement with previously reported LTC-IC characteristics. 
      In conclusion, the CAFC assay enumerates LTC-IC using a direct visual endpoint 
      and allows study of LTC-IC heterogeneity with respect to progenitor cell 
      generation per stem cell clone in various haematologic diseases.
FAU - Breems, D A
AU  - Breems DA
AD  - Institute of Hematology, Erasmus University, Rotterdam, The Netherlands.
FAU - Blokland, E A
AU  - Blokland EA
FAU - Neben, S
AU  - Neben S
FAU - Ploemacher, R E
AU  - Ploemacher RE
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PL  - England
TA  - Leukemia
JT  - Leukemia
JID - 8704895
RN  - 0 (Growth Substances)
RN  - U3P01618RT (Fluorouracil)
SB  - IM
MH  - Adult
MH  - Bone Marrow/drug effects
MH  - Bone Marrow Cells
MH  - Cell Cycle
MH  - Cells, Cultured
MH  - Clone Cells
MH  - Colony-Forming Units Assay/methods
MH  - Drug Resistance
MH  - Fluorouracil/pharmacology
MH  - Granulocytes/cytology
MH  - Growth Substances/pharmacology
MH  - Hematopoietic Stem Cells/classification/*cytology/drug effects
MH  - Humans
MH  - Macrophages/cytology
MH  - Reproducibility of Results
MH  - Stromal Cells/cytology
MH  - Time Factors
EDAT- 1994/07/01 00:00
MHDA- 1994/07/01 00:01
CRDT- 1994/07/01 00:00
PHST- 1994/07/01 00:00 [pubmed]
PHST- 1994/07/01 00:01 [medline]
PHST- 1994/07/01 00:00 [entrez]
PST - ppublish
SO  - Leukemia. 1994 Jul;8(7):1095-104.