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Biomedical subjects

L J Murray

Publications and source records attributed to L J Murray.

At least 37 records · Page 2Linked to original sources
PubMed2000

Parental recall of birthweight: a good proxy for recorded birthweight?

Recent evidence suggests potential associations between birthweight and disease in later life. For resource or other reasons recorded birthweight may be unavailable to researchers who have access to uniquely relevant outcome data. The present study examined the validity of parental recall of birthweight. Parents of 1015 males and females aged 12 and 15 years participating in the Young Hearts Study (a cluster random sample of 1015 males and females aged 12 and 15 years from post-primary schools in Northern Ireland) completed a questionnaire which included a question about their child's birthweight. The answer provided was compared with recorded birthweight obtained from archived computerised child health records with a cut-off point for inaccurate reporting set at +/- 227 g (1/2 lb). The influence of social class and weight at birth on accuracy of recall was also determined. A total of 84.8% of parents accurately recalled their child's birthweight to within 227 g. Parents from non-manual occupation social classes recalled birthweight more accurately than those from manual occupation social classes (88.0 vs. 82.6% accurate: chi2 = 4.81, p = 0.03). Parents of low birthweight infants tended to recall their birthweight less accurately than parents of normal weight infants: 76.1% accurate compared to 86.1% accurate: chi2 = 3.54, p = 0.06. Parents of high birthweight infants recalled their birthweight less accurately than parents of normal weight infants: 78.5% accurate: chi2 = 3.94, p = 0.05. In conclusion, parentally recalled birthweight may be a suitable proxy for recorded birthweight for population based research into disease in childhood and adolescence.

Adolescent↗
PubMed1999

CD34+ cells from mobilized peripheral blood retain fetal bone marrow repopulating capacity within the Thy-1+ subset following cell division ex vivo.

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34+ Thy-1+ cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34+ Thy-1+ CFSElo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34+ Thy-1+ cells (a mean of 1/8.4). In contrast, CD34+ cells that had lost Thy-1 expression during culture (CD34+ Thy-1 CFSElo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1-expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34+ Thy-1+ fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34+ Thy-1+ cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1+ cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34+ cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34+ Thy-1+ cells divided under the conditions tested, promoting susceptibility to retroviral transduction.

Animals↗
PubMed1999

Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division.

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.

Antigens, CD34↗
PubMed1999

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells.

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.

Adult↗
PubMed1999

Epidemiology of Chlamydia pneumoniae infection in a randomly selected population in a developed country.

This cross-sectional study of 400 sera from a randomly selected adult population in Northern Ireland, using a microimmunofluorescence assay, demonstrated high overall seropositivity (70%) for IgG Chlamydia pneumoniae antibodies in developed populations. Seropositivity was shown to be unrelated to gender, age or smoking but there was an inverse trend between infection and educational level achieved as a measure of socio-economic status. IgG levels were also higher during the winter months suggesting seasonal variation of Chlamydia pneumoniae infection. The high prevalence of evidence of exposure to Chlamydia pneumoniae as described in this study may have implications for prevention of cardiovascular disease if further evidence conclusively determines that infection with this organism is a risk factor for cardiovascular disease.

Adult↗
PubMed1999

Chlamydia pneumoniae antibodies are associated with an atherogenic lipid profile.

OBJECTIVE: To determine, within a representative population group of men and women, whether alteration of the lipid profile might underlie the reported association between Chlamydia pneumoniae and ischaemic heart disease. DESIGN AND SETTING: Cross sectional survey in an area with a high incidence of ischaemic heart disease. SUBJECTS: 400 randomly selected participants in the World Health Organisation MONICA project's third population survey in Northern Ireland. MAIN OUTCOME MEASURES: Stored sera were examined by microimmunofluorescence for IgG antibodies to C pneumoniae at a dilution of 1 in 64. Mean total and high density lipoprotein (HDL) cholesterol were compared between seropositive and seronegative individuals with adjustment for age, measures of socioeconomic status, smoking habit, alcohol consumption, body mass index, and the season during which blood had been taken. RESULTS: In seropositive men, adjusted mean serum total cholesterol and HDL cholesterol were 0.5 mmol/l (9.2%) higher and 0.11 mmol/l (9.3%) lower, respectively, than in seronegative men. Differences in women did not achieve statistical significance, but both total cholesterol and HDL cholesterol were higher (3.6% and 5.8%, respectively) in seropositive than in seronegative individuals. CONCLUSIONS: There is serological evidence that C pneumoniae infection is associated with an atherogenic lipid profile in men. Altered lipid levels may underlie the association between C pneumoniae and ischaemic heart disease.

Adult↗
PubMed1998

Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy-1+Lin- cells with preserved ability to engraft SCID-hu bone.

CD34(+)Thy-1(+)Lin- cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34(+)Thy-1(+)Lin- cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34(+)Thy-1(+)Lin- cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34(hi) PKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34(+)Thy-1(+)Lin- cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34(hi) PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34(hi) PKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34(+)Thy-1(+)Lin- cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.

Animals↗
PubMed1998

Thrombopoietin mobilizes CD34+ cell subsets into peripheral blood and expands multilineage progenitors in bone marrow of cancer patients with normal hematopoiesis.

Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, also mediates biologic effects in vitro on hematopoietic cells more primitive than those committed to the megakaryocyte (MK) lineage. To assess the spectrum of hematopoietic effects of recombinant human (rh)TPO in vivo, we evaluated its proliferative effect on bone marrow (BM) progenitor cells, its maturation effect on BM MKs, and its mobilizing effect on peripheral blood (PB) progenitor cells during a phase I clinical laboratory investigation in which rhTPO was administered to cancer patients with normal hematopoiesis. Twelve patients received a single dose of rhTPO (0.3, 0.6, 1.2, or 2.4 microg/kg of body weight) prior to chemotherapy. BM and PB samples from these patients were analyzed 1 to 2 days before (baseline) and 7 days after rhTPO administration. At higher doses (1.2-2.4 microg/kg), rhTPO produced increased concentrations of primitive CD34+Thy-1+Lin-cells (mean 2.1-fold), CD34+mpl+ cells (mean 5.2-fold), CD34+CD41+CD14- promegakaryoblasts (mean 2.9-fold), and myeloerythroid colony-forming cells (mean threefold) in BM. No significant increases in the frequency of BM colony-forming unit (CFU)-MK were observed. Elevated numbers of both immature (2N-8N) and more mature (64N and 128N) CD41+ MKs were detected in BM, with modal ploidy remaining at 16N. Higher doses of rhTPO (1.2-2.4 microg/kg) also induced increased concentrations of CD34+ cell subsets in PB, including both primitive CD34+Thy-1+Lin- (mean 8.8-fold) and MK lineage-committed CD34+CD41+CD14- cells (mean 14.6-fold) as well as various myeloerythroid colony-forming cells (mean 3.6- to 5.5-fold). These results demonstrate that rhTPO given as a single dose not only promotes proliferation and maturation of cells of the MK lineage, but also expands the pool of BM primitive hematopoietic cells. In addition, rhTPO induces mobilization of hematopoietic progenitors into peripheral circulation. The extent to which such multilineage effects on human progenitor cells will contribute to clinical efficacy remains to be determined.

Adolescent↗
PubMed1997

A family of cytokine-inducible inhibitors of signalling.

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Amino Acid Sequence↗
PubMed1997

Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer.

BACKGROUND: Thrombocytopenia is frequently encountered in patients with cancer. It is associated with an increased risk for clinically important bleeding episodes, which increases the demand for platelet transfusion. OBJECTIVE: To assess hematopoietic response to and clinical tolerance of recombinant human thrombopoietin, a recently cloned novel cytokine. DESIGN: Phase I and II clinical cohort study. SETTING: The University of Texas M.D. Anderson Cancer Center, Houston, Texas. PATIENTS: 12 patients with sarcoma who had high risk for severe chemotherapy-induced thrombocytopenia. INTERVENTION: A single intravenous dose of thrombopoietin (0.3 to 2.4 micrograms/kg of body weight) 3 weeks before chemotherapy. MEASUREMENTS: Peripheral blood and bone marrow evaluation before and after thrombopoietin administration. RESULTS: A single dose of thrombopoietin was associated with an increase in platelet counts (mean increase from baseline, 61% to 213%; P = 0.002) in a dose-related manner. This increase began by day 4 in most patients and peaked on a median of day 12. This sustained response was associated with a prolonged serum thrombopoietin half life (20 to 30 hours). The platelets appeared morphologically normal and showed normal aggregation in response to various agonists. Platelet response was accompanied by a dose-related increase in bone marrow megakaryocytes (as much as 4-fold); the expansion of the bone marrow progenitors of myeloid, erythroid, multipotential, and megakaryocytic lineages; and the marked mobilization of progenitors (maximum, 5.7-fold to 10-fold) of multiple cell lineages in the peripheral blood. Treatment was well tolerated, and no serious adverse events occurred. CONCLUSIONS: Thrombopoietin, administered as a single dose, is a potent stimulus for prolonged platelet production in humans. It merits further evaluation for the prevention and treatment of thrombocytopenia.

Antibodies↗
PubMed1997

Epidemiology of Helicobacter pylori infection among 4742 randomly selected subjects from Northern Ireland.

BACKGROUND: Despite the widespread prevalence and serious clinical sequelae of infection with Helicobacter pylori, there have been few large population-based studies, using randomly selected subjects, examining the epidemiology of this infection. AIM: To examine the distribution and determinants of H. pylori infection in a developed country. SUBJECTS AND SETTING: Overall 4742 subjects, aged 12-64, from Northern Ireland were randomly selected. METHODS: Helicobacter pylori specific IgG antibodies were measured by enzyme linked immunosorbent assay, using an acid-glycine extract antigen, in stored serum from subjects who had participated in three linked population-based surveys of cardiovascular risk factors performed in 1986 and 1987. RESULTS: The overall prevalence of H. pylori infection was 50.5%. Prevalence increased with age from 23.4% in 12-14 year olds to 72.7% in 60-64 year olds: chi 2 for trend 518, P < 10(-4). In subjects aged > or = 25, infection was more common in males (60.9%) than females (55.2%): chi 2 = 9.53, P < 0.01. This relation remained significant after adjusting for age, and measures of socioeconomic class: odds ratio (OR) for infection, male versus female was 1.19 (95% confidence interval [CI]: 1.02-1.40). Infection was associated with social class: the adjusted OR of infection in subjects from manual social classes relative to those from non-manual classes was 1.7 (95% CI: 1.47-1.98). Infection was significantly more common in current smokers and ex-smokers than in subjects who had never smoked: adjusted OR for infection, ex-smokers versus never smoked was 1.22 (95% CI: 1.01-1.49); for smokers of > or = 20/day versus never smoked OR = 1.33 (95% CI: 1.05-1.67). Infection was not associated with height in adult males but mean height in infected women was lower than in uninfected women after adjusting for age and socioeconomic status: difference in mean height (SE), -0.85 cm (0.32), P < 0.01. There was no demonstrable relationship between H. pylori infection and current alcohol intake. CONCLUSIONS: This study demonstrated a high prevalence of infection in a population from a developed country. Previously reported associations between H. pylori infection, age, sex, social class, and reduced height in females were confirmed and smoking was identified as a possible risk factor for H. pylori infection.

Adolescent↗
PubMed1996

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells.

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy-1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony-forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.

Adult↗
PubMed1996

Helicobacter pylori infection in children: relation with current household living conditions.

BACKGROUND: Studies demonstrating that deprived household living conditions during childhood are risk factors for acquisition of Helicobacter pylori infection have been performed mainly in adults, who probably acquired the infection several decades ago. This study investigates whether deprived household living conditions remain important risk factors for infection in subjects (children) with recently acquired infection. AIMS: To examine the relation between current household living conditions and acquisition of H pylori infection in childhood. SUBJECTS/SETTING: Opportunistically recruited group of 367 children, aged 3 to 15 years, undergoing routine non-gastrointestinal day surgery. METHODS: Anti-H pylori IgG antibodies measured by a commercial enzyme linked immunosorbent assay validated for use in children. Postal questionnaire collecting sociodemographic data and data on household living conditions. RESULTS: Infection was associated with social class and overcrowding in the household. After adjustment for age, social class, and household density, a positive association remained between infection with H pylori and bed-sharing between children and parents on one or two nights per week, odds ratio for infection (95% CI), 2.29 (1.21, 4.32) or more frequently, odds ratio for infection (95% CI), 2.95 (1.35, 6.45). CONCLUSIONS: The continuing importance of household living conditions in the acquisition of H pylori infection is confirmed and household crowding and sharing a bed with a parent are identified as risk factors for infection.

Adolescent↗
PubMed1996

CD34+Thy-1+Lin- stem cells from mobilized peripheral blood.

Over the last ten years there has been increasing use of mobilized peripheral blood (MPB) progenitor cells as grafts for autologous transplantation. Among the cells comprising these MPB autografts is a subpopulation of CD34+Thy-1+Lineage (Lin)- cells, which is enriched for hematopoietic stem cell (HSC) activity. The percentage of CD34+ cells which express Thy-1 is higher in some samples of MPB than in bone marrow (BM). Using myeloid and erythroid cell depletion prior to high speed cell sorting, it is possible to purify sufficient numbers of CD34+Thy-1+Lin-HSCs from a MPB leukapheresis sample for use as an autograft. CD34+Thy-1+Lin-cells will potentially provide a tumor-depleted autograft for cancer patients. This HSC population is also being studied as a potential target for gene transfer for the treatment of patients with HIV, cancer and a variety of genetic disorders.

Animals↗
PubMed1996

Detection of a primitive megakaryocyte progenitor cell in human fetal bone marrow.

Ontogeny-related changes in megakaryocyte (MK) progenitor cells were a analyzed to further define the cellular hierarchy occurring during human MK development. CD34+ cells were selected from human low-density adult bone marrow (ABM) or unfractionated fetal bone marrow (FBM) and assayed for MK colony formation in a serum-depleted fibrin clot assay system. At days 3, 7, 12, 16, 21, and 28 of incubation, MK colonies were analyzed for colony number, size and number of foci of development. Unifocal CFU-MK-derived colonies cloned from FBM formed after fewer days of in vitro culture and were 2.6-fold larger than those colonies cloned from ABM, However, the frequency of CFU-MK derived colonies cloned from ABM was significantly greater. the MK colonies cloned from FBM morphologically consisted of both pure MK colonies and mixed MK-containing colonies, in which a core of CD41- cells were surrounded by CD41+MKs. Large colonies resembling the primitive burst-forming unit-MK (BFU-MK) also were assayable from both FBM and ABM. These BFU-MK-derived colonies appeared after fewer days of incubation when FBM was assayed compared to ABM, but at a significantly lower frequency. In addition, large unifocal MK colonies consisting of >300 cells (300-1000) appeared from cells cloned from FBM but not ABM. This type of colony, which appears to represent a unique type of MK progenitor cell, was termed a high proliferative potential cell-MK (HPPC-MK). such colonies represent a relatively rare MK progenitor. We conclude that there are a significant ontologic changes in human MK progenitor cell development. The kinetics of MK colony formation from ABM differs significantly from that of FBM. Furthermore, the proliferative capacity of fetal MK progenitor cells appear to be far greater than that of ABM. In addition, a more primitive lineage-restricted MK progenitor cell with extensive proliferative capacity, the HPPC-MK, was assayable exclusively from FBM.

Adult↗
PubMed1996

Fetal bone marrow CD34+CD41+ cells are enriched for multipotent hematopoietic progenitors, but not for pluripotent stem cells.

We have investigated the expression of CD41a (gpIIbIIIa) on a subpopulation of human fetal bone marrow (FBM) CD34+ progenitor cells. Human FBM CD34+Lin- cells were subfractionated into CD41a+ and CD41a- subpopulations by flow cytometry. All the megakaryocyte colony-forming cells (CFU-MK) and almost all the burst-forming units-megakaryocyte (BFU-MK) were found within the CD41a+ subpopulation. In addition, a 14-fold greater number of granulocyte-macrophage colony-forming units (CFU-GM) and a five-fold greater number of mixed lineage progenitor cells (CFU-mix) were observed within the CD34+Lin-CD41a+ subpopulation compared to the CD34+Lin-CD41a- subpopulation. The high proliferative potential of CD34+Lin-CD41a+ cells was demonstrated by their capacity to expand in in vitro culture containing human plasma and recombinant Mpl ligand (thrombopoietin [Tpo]) with production of over 80% CD41b+ (gpIIb+) MKs. However, in long-term bone marrow cultures, the CD34+Lin-CD41a- population contained a significantly higher frequency of cobblestone area-forming cells (CAFC) than the CD34+Lin-CD41a+ population, indicating the presence of a primitive hematopoietic stem cell (HSC) population within the CD34+Lin-CD41a- subset. These data suggest that fetal CD34+Lin-CD41a+ cells are enriched for MK progenitor cells (CFU-MK and BFU-MK), myeloid progenitors, and CFU-mix but do not contain the more primitive CAFC.

Antigens, CD34↗
PubMed1996

Identification of CD34+ subsets after glycoprotease selection: engraftment of CD34+Thy-1+Lin- stem cells in fetal sheep.

Epitopes on the CD34 molecule detected by some CD34 antibodies can be cleaved by a unique glycoprotease from Pasteurella haemolytica, which cleaves only glycoproteins rich in O-linked glycans. A method to isolate CD34+ cells from adult bone marrow was developed subsequently, in which CD34+ cells were isolated in high purity and yield following immunomagnetic bead selection and detachment with the glycoprotease. Using a variety of other cell-surface markers shown here to be insensitive to glycoprotease, committed progenitors of T lymphoid, B lymphoid, monomyeloid, megakaryoblastic, or erythroid lineages could be identified. Significantly, candidate hematopoietic stem cells (HSC) that are contained within a CD34+Lin- (CD2-, CD14-, CD15-, CD16-, CD19-) (or CD34+CD38-) subset expressing the Thy-1 antigen (CDw90), c-kit receptor (CD117), and CDw109 but lacking expression of CD71 and HLA-DR antigens also were detected. Functionally distinct subsets of glycoprotease-selected CD34+ cells were identified and subfractionated using flow cytometry and fluorescence-activated cell sorting (FACS). These subsets included candidate HSCs expressing the CD34+Thy-1+Lin- phenotype, which were sorted from a CD34+ fraction of a mobilized peripheral blood (MPB) sample. In a fetal sheep model, when CD34+Thy-1+Lin- cells were injected intraperitoneally, they were capable of homing to the marrow, where they generated long-term multilineage hematopoiesis and maintained human CD34+ cells, indicating that candidate HSC subsets of CD34+ cells selected with this highly specific enzyme were capable of engraftment in vivo. The ability to identify and purify virtually any phenotypically defined subset of glycoprotease-selected CD34+ stem/progenitor cells should facilitate the study of hematopoiesis in vitro and in animal models in vivo as well as the development of novel genetic techniques for the correction of specific blood cell disorders in humans.

Adult↗