A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia

We examined the stem cell compartment of patients with acquired aplastic anemia (AA) using the long-term culture-initiating cell assay (LTC-IC), in parallel with measurements of CD34+ cells and mature hematopoietic progenitors. Secondary colonies from cells surviving 5 weeks of long-term bone marrow culture (LTBMC) were determined for the peripheral blood (PB) of 68 AA patients and 13 normal controls and for BM of 49 AA patients and 14 controls; because of low cell numbers, formal limiting dilution analysis could only be performed in 10 patients. The relationship of cell input in LTBMC and the output of secondary colonies was linear, allowing quantification of LTC-IC number from bulk cultures. Secondary colony formation was markedly abnormal in severe AA. In contrast to 7.8 colony-forming cells (CFC)/10(5) mononuclear cells in normal BM and 0.14 CFC/10(5) normal PB mononuclear cells, patients with severe disease showed 0.024 CFC/10(5) in BM and 0.0068 CFC/10(5) in PB. Under limiting dilution conditions, patients' cells also showed markedly lower colony-forming ability. In contrast to 4.3 +/- 1 colonies/normal LTC-IC, we obtained only 1.27 +/- 0.09 and 2.0 +/- 0.35 colonies from BM of acute and recovered cases, respectively. These values were used to extrapolate LTC-IC numbers from secondary colony formation in suspension cultures. In PB, calculated LTC-IC were decreased 7.4-fold in new and relapsed severe AA and 2.8- fold in recovered AA. In BM, LTC-IC were decreased 10-fold in new and relapsed AA and sixfold in recovered cases. Compared with measurements obtained on presentation, LTC-IC were lower in post-treatment samples from patients who had failed to recover after intensive immunosuppression and relatively higher in cases at relapse. In recovered patients, LTC-IC number increased but remained below the normal range in 20 of 25. In patients studied serially for 3 to 12 months after treatment, LTC-IC numbers remained stable but low. LTC-IC number correlated with concurrently determined CD34+ cell number and primary hematopoietic colony formation. These results indicate that stem cell numbers, as quantitated by the LTC-IC assay, are markedly diminished in number in all severe AA. Additionally, the function of the stem cell or the stem cell compartment in AA is also abnormal, as inferred from the low clonogenic potential in secondary colony assays. Early hematologic improvement in some patients occurs without increasing numbers of LTC-IC, and a minority of recovered cases show apparent repopulation of the LTC-IC compartment years after treatment.

Download Full-text

Sustained long-term hematologic recovery despite a marked quantitative defect in the stem cell compartment of patients with aplastic anemia after immunosuppressive therapy

American Journal of Hematology ◽

10.1002/1096-8652(200010)65:2<123::aid-ajh6>3.0.co;2-m ◽

2000 ◽

Vol 65 (2) ◽

pp. 123-131 ◽

Cited By ~ 21

Author(s):

J.P. Maciejewski ◽

S. Kim ◽

E. Sloand ◽

C. Selleri ◽

N.S. Young

Keyword(s):

Stem Cell ◽

Aplastic Anemia ◽

Immunosuppressive Therapy ◽

Cell Compartment ◽

Stem Cell Compartment

Download Full-text

Pluripotential stem cell differentiation in hemopoietic colonies

Blood ◽

10.1182/blood.v47.2.315.315 ◽

1976 ◽

Vol 47 (2) ◽

pp. 315-323 ◽

Cited By ~ 7

Author(s):

RL DeGowin ◽

DP Gibson

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Mononuclear Cells ◽

Tritiated Thymidine ◽

Myeloid Cells ◽

Stem Cell Differentiation ◽

Lymphoid Cells ◽

Cell Compartment ◽

Split Dose ◽

Stem Cell Compartment

Abstract To determine if mononuclear cells proliferating in murine hemopoietic spleen colonies were pluripotential in addition to possessing kinetic features of stem cells, we performed sequential studies of mice during their recovery from a split-dose irradiation regimen of 850 roentgens leg shielded-3-hr interval-850 roentgens leg irradiated (850R L.S. 3- L.I.). Injecting tritiated thymidine during stem cell compartment repletion 3 and 4 days after 850R L.S. 3- L.I. resulted in heavily labeled mononuclear cells resembling medium to large leptochromatic lymphocytes in the portion of spleen removed an hour after injection. The splenic remnant obtained from the same mouse 24–48 hr later contained lightly labeled erythroblasts, myeloid cells, and lymphoid cells. Grain counts suggested that erythroblasts and their precursors had undergone about four divisions, myeloid cells and their precursors two to three divisions, and lymphoid cells and their precursors two to three divisions during the 48-hr period. Similar studies in plethoric mice demonstrated the labeling of mononuclear cells on day 4 and their differentiation to myeloid and lymphoid cells by day 6. This finding confirmed that the labeled mononuclear cells were not exclusively erythroblast progenitors. On the basis of these and previous studies of post-irradiation survival and erythropoietic recovery, we conclude that these endogenous monomuclear cells, which resemble medium to large leptochromatic lymphocytes and replicate during stem cell compartment repletion, are pluripotential hemopoietic stem cells.

Download Full-text

Long-term Residual Radiation Effect in the Murine Hematopoietic Stem Cell Compartment

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1985.tb20829.x ◽

1985 ◽

Vol 459 (1 Hematopoietic) ◽

pp. 221-231 ◽

Cited By ~ 4

Author(s):

K.-H. V. WANGENHEIM ◽

H.P, PETERSON, ◽

G. E. HÜBNER ◽

L. E. FEINENDEGEN

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Radiation Effect ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Murine Hematopoietic Stem Cell

Download Full-text

SNP Karyotyping Can Detect Clonality of Stem Cell Compartment and Early Malignant Evolution in Aplastic Anemia

Blood ◽

10.1182/blood.v112.11.443.443 ◽

2008 ◽

Vol 112 (11) ◽

pp. 443-443

Author(s):

Marcin Wlodarski ◽

Sanjay Mohan ◽

Lukasz Gondek ◽

Jungwon Huh ◽

Michael Clemente ◽

...

Keyword(s):

Stem Cell ◽

Aplastic Anemia ◽

Chromosomal Abnormalities ◽

Snp Array ◽

Uniparental Disomy ◽

Multiple Time ◽

Cell Compartment ◽

Monosomy 7 ◽

Stem Cell Compartment ◽

Trisomy 12

Abstract Aplastic anemia (AA) is characterized by pancytopenia due to contraction/destruction of stem cell compartment. Most investigators consider the presence of clonal cytogenetic abnormalities as incompatible with the AA diagnosis. Despite excellent response rates to immunosuppression (IS) in the majority of AA patients, clonal malignant evolution to myelodysplasia (MDS) can occur in 10–15% in 10 years. Among such cases monosomy 7 (mono7) is the most commonly reported cytogenetic abnormality. Routine metaphase cytogenetics (MC) depends upon high numbers of dividing cells with inducible mitosis and is therefore often noninformative in AA. The inability to early detect AA patients at risk for clonal evolution constitutes a significant clinical problem. We hypothesized that high resolution SNP-array technology (SNPA) that allows for the analysis of interphase genomes will improve detection of chromosomal abnormalities in MDS evolving from AA. In addition to MC, we applied Affymetrix chips to study whole genomes of AA patients (N=100; 69 and 67 were investigated using 50K/250K and 6.0 arrays, respectively; 25 patients were studied at multiple time points prior and post IS). Data was analyzed using CNAG v3.0 and Genotyping Console v2.0 and unbalanced lesions were detected, including regions of genomic gain, loss and copy number neutral loss of heterozygozity. Clonal malignant evolution was observed in 13 patients resulting in a conversion rate of 13%. We focused on longitudinal analysis of these patients. Abnormal endpoint MC was detected in a total of 69% of transformed patients (mono7 in 8/13, t(10;18)(q11.2;q21) in 1/13 and trisomy 12 in 1/13 patients, respectively). Remarkably in 5/13 (38%) of evolving AA patients, numerical aberrations were detected earlier by SNPA than MC. Mono7 (N=4) and trisomy 12 (N=1) were detected by SNPA in aspirates that showed normal or noninformative MC, while in subsequent analysis bone marrow exams concurred with the early diagnosis by SNPA. In 3 patients, MC and SNPA results were concordant and in 1 patient SNPA failed to early identify mono7,. Acquired uniparental disomy of various chromosomes was detected in a total of 4 patients and the analysis of nonmyeloid CD3+ cells revealed somatic nature of these lesions: 7q31.31–31.33(4.9mb), 17q11.2-qter(56mb), 6p12.1-pter(56mb) and 3q12.2-qter(97mb). Interestingly, 2 patients with UPD had normal concurrent MC and the clonal lesions were detected before clinical diagnosis of MDS was established. It is likely that depletion of stem cell compartment might favor detection of pseudoclonality manifested as non-pathogenic random chromosomal lesions. In our cohort, in 2 AA patients with normal MC, SNPA analysis prior to IS therapy revealed clonal UPD as confirmed by comparison of myeloid lineage with germ line configuration in CD3+ cells. These lesions disappeared post IS in both patients and nearly 2 years later a newly recruited pathogenic clone with mono7 was detected as a sole abnormality by SNPA and MC. These patients are very illustrative as they may point towards genomic instability as well as depletion of available stem cell pool resulting in frequent recruitment of defective SC. Our study demonstrates that SNPA is a powerful tool to early identify AA patients harboring clonal defects consistent with the diagnosis of MDS, and thus it potentially might have strong clinical implications.

Download Full-text

The kinetics of murine hematopoietic stem cells in vivo in response to prolonged increased mature blood cell production induced by granulocyte colony-stimulating factor

Blood ◽

10.1182/blood.v86.8.2986.2986 ◽

1995 ◽

Vol 86 (8) ◽

pp. 2986-2992 ◽

Cited By ~ 26

Author(s):

G de Haan ◽

B Dontje ◽

C Engel ◽

M Loeffler ◽

W Nijhof

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Blood Cells ◽

Granulocyte Colony Stimulating Factor ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Cell Numbers ◽

Stem Cell Compartment ◽

Stimulating Factor

Abstract Because of the complexity of appropriate stem cell assays, little information on the in vivo regulation of murine stem cell biology or stemmatopoiesis is available. It is unknown whether and how in vivo the primitive hematopoietic stem cell compartment is affected during a continued increased production of mature blood cells. In this study, we present data showing that prolonged (3 weeks) administration of granulocyte colony-stimulating factor (G-CSF), which is a major regulator of mature granulocyte production, has a substantial impact on both the size and the location of various stem cell subset pools in mice. We have used the novel cobblestone area forming cell (CAFC) assay to assess the effects of G-CSF on the stem cell compartment (CAFC days 7, 14, 21, and 28). In marrow, in which normally 99% of the total number of stem cells can be found, G-CSF induced a severe depletion of particularly the most primitive stem cells to 5% to 10% of normal values. The response after 7 days of G-CSF treatment was an increased amplification between CAFC day 14 and 7. However, this response occurred at the expense of the number of CAFC day 14. It is likely that the resulting gap of CAFC day 14 cell numbers was subsequently replenished from the more primitive CAFC day 21 and 28 compartments, because these cell numbers remained low during the entire treatment period. In the spleen, the number of stem cells increased, likely caused by a migration from the marrow via the blood, leading to an accumulation in the spleen. The increased number of stem cells in the spleen overcompensated for the loss in the marrow. When total body (marrow and spleen) stem cell numbers were calculated, it appeared that a continued increased production of mature granulocytes resulted in the establishment of a higher, new steady state of the stem cell compartment; most committed stem cells (CAFC day 7) were increased threefold, CAFC day 14 were increased 2.3-fold, CAFC-day 21 were increased 1.8-fold, and the most primitive stem cells evaluated, CAFC day 28, were not different from normal, although now 95% of these cells were located in the spleen. Four weeks after discontinuation of the G-CSF treatment, the stem cell reserve in the spleen had returned to a normal level, whereas stem cell numbers in marrow had recovered to values above normal. This study shows that the primitive stem cell compartment is seriously perturbed during an increased stimulation of the production of mature blood cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

Obesity alters the long-term fitness of the hematopoietic stem cell compartment through modulation of Gfi1 expression

Journal of Experimental Medicine ◽

10.1084/jem.20170690 ◽

2017 ◽

Vol 215 (2) ◽

pp. 627-644 ◽

Cited By ~ 27

Author(s):

Jung-Mi Lee ◽

Vinothini Govindarajah ◽

Bryan Goddard ◽

Ashwini Hinge ◽

David E. Muench ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Normal Environment ◽

Key Driver ◽

The Impact ◽

Insight Into

Obesity is a chronic organismal stress that disrupts multiple systemic and tissue-specific functions. In this study, we describe the impact of obesity on the activity of the hematopoietic stem cell (HSC) compartment. We show that obesity alters the composition of the HSC compartment and its activity in response to hematopoietic stress. The impact of obesity on HSC function is progressively acquired but persists after weight loss or transplantation into a normal environment. Mechanistically, we establish that the oxidative stress induced by obesity dysregulates the expression of the transcription factor Gfi1 and that increased Gfi1 expression is required for the abnormal HSC function induced by obesity. These results demonstrate that obesity produces durable changes in HSC function and phenotype and that elevation of Gfi1 expression in response to the oxidative environment is a key driver of the altered HSC properties observed in obesity. Altogether, these data provide phenotypic and mechanistic insight into durable hematopoietic dysregulations resulting from obesity.

Download Full-text

PR-domain–containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function

Blood ◽

10.1182/blood-2011-02-334680 ◽

2011 ◽

Vol 118 (14) ◽

pp. 3853-3861 ◽

Cited By ~ 57

Author(s):

Yi Zhang ◽

Sandra Stehling-Sun ◽

Kimberly Lezon-Geyda ◽

Subhash C. Juneja ◽

Lucie Coillard ◽

...

Keyword(s):

Stem Cell ◽

Cell Function ◽

Critical Role ◽

Growth Control ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Pr Domain

Abstract The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed MEm1, which eliminates Mds1-Evi1 (ME), the longer, PR-domain–containing isoform produced by the gene (also known as PRDM3). β-galactosidase–marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin−Sca-1+c-Kit+ cells (LSKs) of MEm1 documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into MEm1 LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain–containing ME in linking p57-kip2 regulation to long-term HSC function.

Download Full-text

Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture

Blood ◽

10.1182/blood.v80.9.2237.bloodjournal8092237 ◽

1992 ◽

Vol 80 (9) ◽

pp. 2237-2245 ◽

Cited By ~ 1

Author(s):

P Valent ◽

E Spanblochl ◽

WR Sperr ◽

C Sillaber ◽

KM Zsebo ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mast Cells ◽

Peripheral Blood ◽

Stem Cell Factor ◽

Mononuclear Cells ◽

Precursor Cells ◽

Control Medium ◽

Long Term Culture

In the murine system, a number of cytokines (including interleukin-3 [IL-3], IL-4, and stem cell factor [SCF]) promote the growth of mast cells (MCs). However, so far little is known about factors controlling differentiation of human MCs. Recent data suggest that human MCs express receptors (R) for SCF. The aim of the present study was to investigate whether recombinant human (rh) SCF induces differentiation of human MCs from their precursor cells. For this purpose, bone marrow (BM; normal donors, n = 6) and peripheral blood (PB; normal donors, n = 11) mononuclear cells (MNC) were cultured in the presence of rhSCF, rhIL-3, rhIL-4, rhIL-9, recombinant human macrophage colony-stimulating factor (rhM-CSF), or control medium in long-term (8 weeks) suspension cultures. After 4 weeks, up to 5% of the MNC (BM and PB) cultured in the presence of rhSCF, but not in the presence of other cytokines, were found to exhibit the characteristics of MCs. These MCs expressed the YB5.B8-reactive domain of the SCF R as well as IgE R, as determined by combined toluidine blue/immunofluorescence staining. Myeloid antigens, likewise expressed on human basophils (ie, CD11b, CDw65, and Bsp-1), could not be detected on these cells. Furthermore, rhSCF, but not rhIL- 3, rhIL-4, rhIL-9, or rhM-CSF, induced dose- and time-dependent increases in the formation of cellular tryptase (an MC-specific enzyme) (rhSCF [100 ng/mL], 1,308 +/- 679 ng/mL v control medium, 18 +/- 6 ng/mL tryptase on day 35 of PB cell cultures), as well as an increase in cellular histamine. After 6 to 8 weeks, when other mature hematopoietic cells decreased, MCs still could be detected in culture, with up to 40% of all cells being MCs. To test whether rhSCF also activates tissue MCs, we performed histamine release experiments (dispersed tissue; lung, n = 3; uterus, n = 3). SCF was found to enhance (by up to 3.4-fold) the capacity of the MCs to release histamine upon cross-linkage of IgE R with anti-IgE. Together, these observations suggest that rhSCF induces in vitro differentiation of human MCs from their BM and PB precursor cells in long-term culture and upregulates MC releasability.

Download Full-text