A Novel Nr4a1GFP Reporter System Reveals That Nr4a1 Expression Identifies Long-Term Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2339-2339
Author(s):  
Ruben Land ◽  
Trevor Barlowe ◽  
Shwetha Manjunath ◽  
Sophie Eiger ◽  
Matthew Gross ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Reporter System ◽  
Hematopoietic Stem ◽  
And Function

Abstract Abstract 2339 Recent studies have highlighted the importance of the NR4A nuclear receptor family (Nur77 (Nr4a1), Nurr1 (Nr4a3), Nor1 (Nr4a2)) in the regulation of hematopoiesis. In murine models, NR4A gene deficiencies lead to aberrant proliferation of hematopoietic stem cells, and can lead to acute myeloid leukemia (AML). NR4A gene deficiencies also appear to be a feature in human AML cells. In order to better understand the pattern of expression and function of NR4A family members during normal hematopoiesis, we have developed a novel reporter mouse where the Nr4a1 promoter drives GFP expression (Nr4a1GFP). Our analyses reveal a hierarchy in Nr4a1 expression among bone marrow hematopoietic stem cells: long-term (LT) HSC's (CD150+CD48-LSKs) express the highest levels of Nr4a1GFP, more mature HSC's and multilineage progenitor populations (CD150+CD48+ and CD150-CD48+ LSKs) express intermediate levels, and common myeloid progenitors (CMLs, defined as Lin-c-kit+sca-1-) express no Nr4a1GFP. Interestingly, circulating LSK's in the spleen express Nr4a1GFP at higher levels than their bone marrow counterparts. In support of data suggesting that Nr4a family members regulate quiescence, we find that 1) all hematopoietic stem cells that remain in the bone marrow after acute (36h) 5-FU treatment express Nr4a1GFP, 2) Nr4a1GFP expression decreases among circulating splenic LSKs 48 hours after treatment with PolyI:C, and 3) Nr4a1GFP expression increases markedly when stem cells are cultured in vitro under conditions that promote quiescence. We will use this novel system to more directly address the role of Nr4a1 expression in hematopoiesis by evaluating the cell cycle status and defining the reconstitution potential of HSC's on the basis of their Nr4a1GFP expression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The chemokine GROβ mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 860-869 ◽  
Author(s):  
Seiji Fukuda ◽  
Huimin Bian ◽  
Andrew G. King ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
And Function ◽  
Stimulating Factor ◽  
Cxcr4 Axis

Abstract Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GROβ (GROβΔ4/CXCL2Δ4) or the combination of GROβΔ4 plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF–mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GROβ-mobilized grafts. PBSCs mobilized by GROβΔ4 alone or with G-CSF contained significantly more Sca-1+-c-kit+-lineage− (SKL) cells and more primitive CD34−-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GROβΔ4-mobilized SKL cells adhered better to VCAM-1+ endothelial cells compared with G-CSF–mobilized cells. GROβΔ4-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1α in vitro that was associated with higher CD26 expression. However, GROβΔ4-mobilized SKL and c-Kit+ lineage− (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GROβΔ4-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GROβΔ4-mobilized cells is less dependent on the SDF-1α/CXCR4 axis.

scholarly journals An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2755-2763 ◽  
Author(s):  
RE Ploemacher ◽  
JP van der Sluijs ◽  
JS Voerman ◽  
NH Brons
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Dilution Assay ◽  
Limiting Dilution Assay ◽  
Colony Forming Units ◽  
Limiting Dilution

We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.

scholarly journals Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3704-3704
Author(s):  
Aldona A Karaczyn ◽  
Edward Jachimowicz ◽  
Jaspreet S Kohli ◽  
Pradeep Sathyanarayana
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

Critical Role Of Jak2 In The Maintenance and Function Of Adult Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1180-1180
Author(s):  
Hajime Akada ◽  
Saeko Akada ◽  
Golam Mohi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Critical Role ◽  
Receptor Protein ◽  
Specific Gene ◽  
Hematopoietic Stem ◽  
And Function

Abstract Hematopoietic stem cells (HSCs) play an essential role in the long-term maintenance of hematopoiesis. Various intracellular signaling proteins, transcription factors and extracellular matrix proteins contribute to the maintenance and function of HSCs. Jak2, a member of the Janus family of non-receptor protein tyrosine kinases, is activated in response to a variety of cytokines. It has been shown that germ-line deletion of Jak2 results in embryonic lethality whereas post-natal or adult stage deletion of Jak2 results in anemia and thrombocytopenia in mice. However, the role of Jak2 in the maintenance and function of adult HSCs has remained elusive. Understanding the normal function of Jak2 in adult HSC/progenitors is of considerable significance since mutations in Jak2 have been associated with several myeloproliferative neoplasms (MPNs), and most patients treated with Jak2 inhibitors exhibit significant hematopoietic toxicities. To assess the role of Jak2 in adult HSCs, we have utilized a conditional Jak2 knock-out (Jak2 floxed) allele and an inducible MxCre line that can efficiently express Cre recombinase in adult HSC/progenitors after injections with polyinosine-polycytosine (pI-pC). We have found that deletion of Jak2 in adult mice results in pancytopenia, bone marrow aplasia and 100% lethality within 25 to 42 days after pI-pC induction. Analysis of the HSC/progenitor compartments revealed that Jak2-deficiency causes marked decrease in long-term HSCs, short-term HSCs, multipotent progenitors and early progenitors of all hematopoietic lineages, indicating a defect at the earliest stage of adult hematopoietic development. We have found that deletion of Jak2 leads to increased HSC cell cycle entry, suggesting that Jak2-deficiency results in loss of quiescence in HSCs. Jak2-deficiency also resulted in significant apoptosis in HSCs. Furthermore Jak2-deficient bone marrow cells were severely defective in reconstituting hematopoiesis in lethally-irradiated recipient animals. Competitive repopulations experiments also show that Jak2 is essential for HSC functional activity. We also have confirmed that the requirement for Jak2 in HSCs is cell-autonomous. To gain insight into the mechanism by which Jak2 controls HSC maintenance and function, we have performed phospho flow analysis on HSC-enriched LSK (lin-Sca-1+c-kit+) cells. TPO and SCF-evoked Akt and Erk activation was significantly reduced in Jak2-deficient LSK compared with control LSK. Stat5 phosphorylation in response to TPO was also completely inhibited in Jak2-deficient LSK cells. In addition, we observed significantly increased intracellular reactive oxygen species (ROS) levels and enhanced activation of p38 MAPK in Jak2-deficient LSK cells, consistent with the loss of quiescence observed in Jak2-deficient HSCs. Treatment with ROS scavenger N-acetyl cysteine partially rescued the defects in Jak2-deficient HSCs in reconstituting hematopoiesis in lethally irradiated recipient animals. Gene expression analysis revealed significant downregulation of HSC-specific gene sets in Jak2-deficient LSK cells. Taken together, our data strongly suggest that Jak2 plays a critical role in the maintenance of quiescence, survival and self-renewal of adult HSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Diabetes impairs the interactions between long-term hematopoietic stem cells and osteopontin-positive cells in the endosteal niche of mouse bone marrow

2013 ◽  
Vol 305 (7) ◽  
pp. C693-C703 ◽  
Author(s):  
Hironori Chiba ◽  
Koji Ataka ◽  
Kousuke Iba ◽  
Kanna Nagaishi ◽  
Toshihiko Yamashita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mouse Bone Marrow ◽  
Diabetic Mice ◽  
Hematopoietic Stem ◽  
Coculture System ◽  
Endosteal Niche

Hematopoietic stem cells (HSCs) are maintained, and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow. Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term HSCs (LT-HSCs) and osteopontin-positive (OPN) cells in endosteal niche. To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and nondiabetic mice. In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, β-catenin, and N-cadherin on LT-HSCs and β1-integrin, β-catenin, angiopoietin-1, and CXCL12 on OPN cells. In an in vitro coculture system, LT-HSCs isolated from nondiabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely, in LT-HSCs derived from diabetic mice exposed to nondiabetic OPN cells, the decreased mRNA expressions of Tie2, β-catenin, and N-cadherin were restored to normal levels. The effects of diabetic or nondiabetic OPN cells on LT-HSCs shown in this coculture system were confirmed by the coinjection of LT-HSCs and OPN cells into bone marrow of irradiated nondiabetic mice. Our results provide new insight into the treatment of diabetes-induced LT-HSC abnormalities and suggest that the replacement of OPN cells may represent a novel treatment strategy.

Murine Hematopoietic Stem Cells Require Akt1 and Akt2 for Long-Term Self-Renewal

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 502-502
Author(s):  
Marisa M. Juntilla ◽  
Vineet Patil ◽  
Rohan Joshi ◽  
Gary A. Koretzky
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Akt Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Murine hematopoietic stem cells (HSCs) rely on components of the Akt signaling pathway, such as FOXO family members and PTEN, for efficient self-renewal and continued survival. However, it is unknown whether Akt is also required for murine HSC function. We hypothesized that Akt would be required for HSC self-renewal, and that the absence of Akt would lead to hematopoietic failure resulting in developmental defects in multiple lineages. To address the effect of Akt loss in HSCs we used competitive and noncompetitive murine fetal liver-bone marrow chimeras. In short-term assays, Akt1−/−Akt2−/− fetal liver cells reconstituted the LSK compartment of an irradiated host as well or better than wildtype cells, although failed to generate wildtype levels of more differentiated cells in multiple lineages. When placed in a competitive environment, Akt1−/−Akt2−/− HSCs were outcompeted by wildtype HSCs in serial bone marrow transplant assays, indicating a requirement for Akt1 and Akt2 in the maintainance of long-term hematopoietic stem cells. Akt1−/−Akt2−/− LSKs tend to remain in the G0 phase of the cell cycle compared to wildtype LSKs, suggesting the failure in serial transplant assays may be due to increased quiesence in the absence of Akt1 and Akt2. Additionally, the intracellular content of reactive oxygen species (ROS) in HSCs is dependent on Akt signaling because Akt1−/−Akt2−/− HSCs have decreased ROS levels. Furthermore, pharmacologic augmentation of ROS in the absence of Akt1 and Akt2 results in an exit from quiescence and rescue of differentiation both in vivo and in vitro. Together, these data implicate Akt1 and Akt2 as critical regulators of long-term HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

The spleen is a major site of megakaryopoiesis following transplantation of murine hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 3975-3982 ◽  
Author(s):  
William B. Slayton ◽  
Ann Georgelas ◽  
L. Jeanne Pierce ◽  
Kojo S. Elenitoba-Johnson ◽  
S. Scott Perry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Stem ◽  
Glucose Phosphate ◽  
Stem Cell Pool

The stem cell pool can be fractionated by using the mitochondrial dye, rhodamine-123, into Rholow hematopoietic stem cells and Rhohigh progenitors. Rholow stem cells permanently engraft all lineages, whereas Rhohighprogenitors transiently produce erythrocytes, without substantial platelet or granulocyte production. We hypothesized that the inability of the Rhohigh cells to produce platelets in vivo was due to the fact that these cells preferentially engraft in the spleen and lack marrow engraftment. Initially, we demonstrated that Rhohigh progenitors produced more megakaryocytes in vitro than Rholow stem cells did. To study the activity of the Rholow and Rhohighsubsets in vivo, we used mice allelic at the hemoglobin and glucose phosphate isomerase loci to track donor-derived erythropoiesis and thrombopoiesis. Rholow stem cells contributed to robust and long-term erythroid and platelet engraftment, whereas Rhohigh progenitors contributed only to transient erythroid engraftment and produced very low numbers of platelets in vivo. Donor-derived megakaryopoiesis occurred at higher densities in the spleen than in the bone marrow in animals receiving Rholowstem cells and peaked around day 28. Blockade of splenic engraftment using pertussis toxin did not affect the peak of splenic megakaryopoiesis, supporting the hypothesis that these megakaryocytes were derived from progenitors that originated in the bone marrow. These data emphasize that in vitro behavior of hematopoietic progenitor cell subsets does not always predict their behavior following transplantation. This study supports a major role for the spleen in thrombopoiesis following engraftment of transplanted stem cells in irradiated mice.

scholarly journals Etoposide-Initiated MLL Rearrangements Detected at High Frequency in Human Primitive Hematopoietic Stem Cells with In Vitro and In Vivo Long-Term Repopulating Potential.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 98-98 ◽  
Author(s):  
Jolanta Libura ◽  
Marueen Ward ◽  
Grzegorz Przybylski ◽  
Christine Richardson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Scid Mouse ◽  
P Value ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Rearrangements involving the MLL gene locus at chromosome band 11q23 are observed in therapy-related acute myeloid leukemia and myelodysplastic syndromes following treatment with topoisomerase II (topoII) inhibitors including etoposide. We have shown that one hour of etoposide exposure (20–50 μM) stimulates stable MLL rearrangements in primary human CD34+ cells and that the spectrum of repair products within MLL gene is broader than so far described (Libura et al, Blood, 2005). Clinical data suggest that MLL-associated malignant leukemias originate within primitive hematopietic stem cells capable of differentiation into all hematopoietic lineages and repopulation of myelo-ablated hosts. These cells can be analyzed using the in vivo NOD-SCID mouse model as well as the in vitro long-term culture initiating cell (LTC-IC) assay. We adopted our in vitro CD34+ cell culture model to investigate the impact of etoposide exposure on the most primitive hematopoietic stem cells using parallel assays for LTC-IC and NOD-SCID Repopulating Cells (SRC). Following etoposide exposure (20–50 μM for 1 hour), and 48–96 hours recovery in vitro, untreated control and etoposide-treated CD34+ cells were either seeded in LTC-IC with a supportive feeder layer (Stem Cell Technologies, Inc.), or injected into NOD-SCID mice (0.1–1.5x106 cells per mouse). After 12 weeks, both LTC-IC cultures and bone marrow cells from NOD-SCID mice were seeded in methylcellulose media supplemented with growth factors that promote only human cell colony formation. An increased number of colonies in etoposide-treated samples was obtained from LTC-IC cultures in 3 out of 5 experiments (p value<0.05). This increase in colony number was more dramatic in etoposide-treated samples from NOD-SCID bone marrow (57 versus 0, 8 versus 0). These data demonstrate that etoposide exposure can significantly alter the potential of early hematopoietic stem cells to survive and proliferate both in vitro and in vivo. Injection of as few as 3x105 CD34+ cells into a NOD-SCID mouse was sufficient to obtain methylcellulose colonies, suggesting that this method can be used for the analysis of cells obtained from a single patient sample. Mutation analysis of human methylcellulose colonies derived from both LTC-IC and NOD-SCID was performed by inverse PCR and ligation-mediated PCR followed by sequencing. This analysis revealed that rearrangements originating within the MLL breakpoint cluster region (bcr) were present in 12 out of 29 colonies from etoposide-treated samples versus 5 out of 39 colonies from control samples (p value <0.01), demonstrating that etoposide exposure promotes stable rearrangements within a hematopoietic stem cell compartment with significant proliferative potential. Eight of the 17 events were sequenced, and showed 6 MLL tandem duplications within intron 8, one complex translocation between MLL and chr.15 and tandem duplication, and one event with foreign sequence of unknown origin. Our data are the first report of the spectrum and frequency of MLL rearrangements following topo II inhibitor exposure in a cell population thought to be the target for recombinogenic events leading to therapy-related leukemias.

scholarly journals An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2755-2763 ◽  
Author(s):  
RE Ploemacher ◽  
JP van der Sluijs ◽  
JS Voerman ◽  
NH Brons
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Dilution Assay ◽  
Limiting Dilution Assay ◽  
Colony Forming Units ◽  
Limiting Dilution

Abstract We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.

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