Long-term erythropoietic repopulating ability of old, young, and fetal stem cells.

It is possible that erythropoietic stem cells do not age. This would mean that stem cells from old donors can function as well as those from young or fetal donors. The competitive repopulation assay has been used to test long-term stem cell function by directly comparing how well competing stem cells repopulate a recipient and produce differentiated cell types. C57BL/6J (B6) mice were used as donors, while recipients and competitors were WBB6F1 hybrids with genetically distinguishable hemoglobin. Lethally irradiated young WBB6F1 recipients were given a mixture of 2.5 X 10(6) cells from B6 old marrow, young marrow, or fetal liver donors; each recipient also received a standard dose of 1 X 10(6) marrow cells from a pool of young WBB6F1 competitors. Surprisingly, the old marrow cells competed the best in repopulating the recipients. This pattern was maintained even after recovery from sublethal irradiation, a treatment that severely stresses stem cells. This stress was demonstrated when sublethal irradiation caused a 20-fold decline in repopulating ability measured using hemoglobin markers, and a 3- to 7-fold decline using chromosome markers. Stem cells from old marrow competed better than young or fetal cells in similar experiments using immunologically crippled recipients or using unirradiated W/Wv recipients that are immunologically intact. In both types of recipients, the advantage of old marrow cells again persisted after recovery from sublethal irradiation. Other genotypes were tested, and marrow cells from old B6CBAF1 donors competed better than those from young donors of that genotype. However, marrow cells from young CBA donors completed better than those from old CBA donors. These results support the hypothesis that stem cells do not age, and suggest that regulatory changes with age promote rapid stem cell repopulation in B6 and B6CBAF1 mice, but inhibit it in CBA mice.

Effects of transplantation on the primitive immunohematopoietic stem cell.

Transplantation has strong deleterious effects on the primitive immunohematopoietic stem cells (PSC) from which circulating lymphocytes and erythrocytes are descended. We studied these effects over 300-400 d, testing whether PSC numbers, repopulating abilities, or both, were reduced. Equivalent PSC numbers were estimated in recipients of mixtures of genetically different cells, using the binomial model with covariance. Percentages of lymphocyte and erythrocyte types were closely correlated, as were percentages of either type sampled at intervals of several months. This suggests that the same PSC produced lymphoid and myeloid cells, and that most circulating cells were descended from the same PSC over hundreds of days. Equivalent PSC concentrations were approximately 1/10(5) fresh marrow cells, and were about twofold lower using previously transplanted marrow. However, such marrow repopulated only one-seventh to one-eighth as well as fresh marrow. Apparently, transplantation not only reduces PSC concentrations, but also reduces the repopulating ability per PSC. This may result from excessive stimuli to differentiate that overbalance the stimuli for PSC to replenish themselves.

Ultimate erythropoietic repopulating abilities of fetal, young adult, and old adult cells compared using repeated irradiation.

Erythropoietic repopulating abilities of fetal liver cells and young and old adult marrow cells were compared as follows: Equal numbers of cells from a donor of each age were mixed with a constant portion of cells pooled from genetically distinguishable competitors. These mixtures were transplanted into stem cell-depleted recipients, and the proportions of recipient hemoglobin that were donor type measured the relative effectiveness of early erythropoietic precursor cells from the various donors (Fig. 1). At intervals of 3-6 mo, recipients were sublethally irradiated, requiring a new round of competitive repopulation. When B6 mice were used as donors, with WBB6F1 competitors and recipients, the highest levels of stem cell activity were found using old donors (Tables I, III). This was true even with unirradiated, immune-competent W/Wv recipients (Table III). When donors and recipients were WBB6F1 hybrids, with B6 competitors, fetal cells initially gave higher levels of repopulating ability, and they were similar to the adult and old marrow cells after 400 d and after recovery from two sublethal irradiations (Table II). These effects were mostly insignificant and probably reflect small differences in initial stem cell concentrations that are brought out by the sensitivity of the competitive repopulation assay. Clearly, ultimate erythropoietic stem cell proliferative capacities did not decline as a result of the proliferation required between 15 d of fetal life and old age. Repopulating abilities of 12-d fetal liver cells were not detectable. We also showed that the proportions of newly synthesized hemoglobins made by the two types of stem cells in tetraparental mice remained nearly constant when tested at 3-d intervals over 30 d. Minimum numbers of stem cells producing erythrocytes over a single 3-d period were calculated as 62 and 128, but these are too low, since variances were similar in the tetraparental mice and in the F1 hybrid control. This contradicts the hypothesis that erythropoietic stem cells reserve limited proliferative capacities by proliferating one or a few at a time. We suggest that erythropoietic stem cells have essentially unlimited proliferative capacities and are found in approximately equal concentrations in the primary erythropoietic organs after 15 or 16 d of fetal life.

Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure.

Long-term functional capacities of marrow cell lines were defined by competitive repopulation, a technique capable of detecting a small decline in repopulating abilities. There was little or no difference between cells from old and young donors, but a single serial transplantation caused a large decline in repopulating ability. Varying the numbers of marrow cells transplanted into the initial carrier from 10(5) to 10(7) did not alter the ability of the carrier's marrow cells to repopulate in competition with previously untransplanted cells. This ability was improved only in carriers that had received 10(8) marrow cells, although deleterious effects of transplantation were still present. These effects were not solely caused by cell damage from the transplantation procedure, because transplantation by parabiosis, or recovery from sublethal irradiation without transplantation, reduced repopulating abilities as much as transplanting 10(5) to 10(7) marrow cells. The transplantation effect also was not caused solely by irradiation, because the same effect appeared in unirradiated W/Wv carriers. The transplantation effect was more pronounced when donors were identified by hemoglobin type than by chromosome markers, implying that nonerythroid cell lines may be less affected by transplantation than erythroid precursor cells. When the effects of a lifetime of normal function and a single transplantation were compared, the latter caused 3-7 times more decline in repopulating abilities of phytohemagglutinin-responsive cell precursors, and at least 10-20 times more decline in erythroid cell precursors. Stem cell lines can be serially transplanted at least five times before losing their ability to repopulate and save lethally irradiated recipients or to cure genetically anemic mice. Therefore, if transplantation causes an acceleration of the normal aging process, these figures suggest that stem cells should be able to function normally through at least 15-50 life spans.

Loss of proliferative capacity in immunohemopoietic stem cells caused by serial transplantation rather than aging.

Marrow stem cell lines from old donors and those from young controls gave equally rapid rates of colony growth on spleens of irradiated mice. Old and young stem cell lines competed equally well with chromosomally marked marrow stem cells from a young donor in producing cell types that are stimulated by bleeding; old cells competed 70% as well as young in producing cell types stimulated by phytohemagglutinin (PHA) in vitro. After a single serial transplantation, the rates of colony growth declined 1.5- to 2.5-fold, and the ability to compete declined 2- to 4-fold for bleeding-stimulated and 4- to 10-fold for PHA-stimulated cells. Thus, immediate stem cell proliferative capacities decline much more after one serial transplantation than after a lifetime of normal function.

Undamped oscillations of pyridine nucleotide and oxygen tension in chemostat cultures of Klebsiella aerogenes.

Klebsiella aerogenes was grown in chemostat culture with the pH controlled to +/-0.01 and temperature to +/-0.1 degrees C. The oxygen tension of the culture was regulated by changing the partial pressure of oxygen in the gas phase and recorded by means of an oxygen electrode. Reduced pyridine nucleotide was monitored continuously in the culture by means of direct fluorimetry. On applying an anaerobic shock to the culture, damped oscillations in pyridine nucleotide fluorescence were obtained. Further anaerobic shocks decreased the damping and eventually gave rise to undamped oscillations of a 2-3 min period which continued for several days. These oscillations were paralleled by oscillations of the same frequency in respiration rate. The amplitude of the oscillations in the respiration rate was equivalent to only 1% of the total steady-state respiration, whereas that of pyridine nucleotide oscillations was equivalent to 10% of the total aerobic/anaerobic fluorescence response. The oscillations ceased on interrupting the glucose feed but restarted on adding excess glucose to the culture. Addition of succinate also restarted the oscillations so that they appear not to be of glycolytic origin. The frequency of oscillations varied with growth rate and conditions. Oscillations of much lower frequency were obtained under limited-oxygen and anaerobic conditions than under fully aerobic conditions. Under glucose-limited conditions, fluctuations were found in adenosine triphosphate (ATP) content which were in phase with the pyridine nucleotide oscillations, but under nitrogen-limited growth conditions no such fluctuations in ATP were observed. The primary oscillating pathway could not be identified but the mechanism would appear to be quite different from that involved in oscillations observed in yeast cells. The synchronization of oscillations and observations of negative damping could be explained by a syntalysis effect.

The appearance of sustained equatorial surface westerlies during the 1982 pacific warm event.

In June 1982 a band of anomalous southerly surface wind, extending from the equator as far south as the Tasman Sea, formed east of Australia (150 degrees E to 160 degrees E). This flow crossed the equator just before the appearance of sustained westerly winds on the equator somewhat west of the date line. Because these westerly winds induced the initial strong equatorial warming of the ocean east of the date line during the 1982 El Niño-Southern Oscillation (ENSO) event, the southerly jet appears to be an important atmospheric component leading to the onset of the vigorous phase of this event. Some historical evidence suggests that anomalous southerly winds in the same region occurred prior to the appearance of sustained equatorial westerly winds in the major ENSO events of 1957, 1965, and 1972.

Zonal winds in the central equatorial pacific and el nino.

Easterly trade winds from near-equatorial islands in the central Pacific weakened before each El Niño between 1950 and 1978, except for the 1963 El Niño. The weakening of the easterlies and their later collapse did not occur uniformly over several months, but rather through a series of strong westerly wind bursts lasting 1 to 3 weeks. The bursts may force equatorial Kelvin waves in the ocean that can both initiate and sustain the sea surface warming characteristics of El Niño events.

Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rt islets of langerhans.

Activation of Ca2+ -calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic beta-cell. To study the properties of suc kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [gamma-32P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca2+ plus calmodulin or by cyclic AMP. The major effect of Ca2+ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53,100 +/- 500 (n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 micrometers free Ca2+ and 0.7 micrometers calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a proteins of similar molecular weight could be enhanced to a lesser extent in the absence of Ca2+ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55,000 and 70-80,000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.

5-Fluorouracil spares hemopoietic stem cells responsible for long-term repopulation.

The long-term immunohemopoietic reconstituting ability of bone marrow, treated with a single administration of 5-fluorouracil (5-FU), was measured to determine whether 5-FU caused any deleterious effect upon primitive stem cells (PSCs). Cells from 5-FU-treated marrow donors were mixed in four different proportions of total marrow contents with untreated competitor marrow containing genetically distinguishable hemoglobin (Hb) and glucosephosphate isomerase (GPI) transplantation markers. These cell mixtures were introduced into lethally irradiated hosts. The functional ability of the donor cell population was assessed by measuring the percentage of donor type Hb and GPI found in the host's circulating erythrocytes and lymphocytes, respectively. Bone marrow from mice treated with 5-FU 1, 5, and 8 days prior to transplantation produced circulating lymphoid and erythroid cells as well as equal fractions of untreated fresh marrow when surveyed approximately 90 days after transplantation. Normal reconstitutive ability was thus maintained despite a tenfold reduction in marrow cell numbers when donor mice had been treated with 5-FU 5 days prior to transplantation. Donor marrow treated with 5-FU 15 days prior to transplantation had slightly decreased repopulating ability in one of two experiments. A second round of repopulation was stimulated subsequent to the initial 90-day screening by giving hosts a sublethal (500 rad) dose of irradiation. After 3-4 months, Hb and GPI parameters were the same as preirradiation values. Thus, the radioresistance of 5-FU treated PSCs remains comparable to that of fresh marrow, and their relative repopulating ability was not comprised by the additional stress of sublethal irradiation. After both rounds of repopulation the myeloid and lymphoid pathways were repopulated equally well by PSCs surviving 5-FU treatment. Repopulation of both pathways to the same extent suggests a common precursor as the proliferative agent. These results indicate that the PSCs were unaffected after a single treatment with 5-FU, although they were concentrated tenfold.

Lymphoid and erythroid repopulation in B6 W-anemic mice: a new unirradiated recipient.

The W-anemic family of mouse mutants is an important model for studying repopulation in unirradiated recipients. This is the first study of blood lymphoid cell repopulation in adult W-anemic mutants given high doses of marrow cells, and it shows a wide difference in repopulation rates of circulating lymphoid and erythroid cells. This study also offers an improved model for marrow transplantation, using W alleles that are spontaneous mutations on the widely used inbred strain C57BL/6J (B6). Unirradiated B6-W41J/W41J or -W41J/W39J recipients of 2 x 10(6) B6 marrow cells are completely repopulated with donor erythrocytes after 3 months, whereas complete repopulation of lymphocytes requires a year. Surprisingly, the eventual degree of repopulation is independent of the severity of the mutation. The new mutants are not as anemic as the commonly used WBB6F1-W/Wv anemic mutants, they have a much higher ability to form macroscopic spleen colonies (spleen colony-forming units, CFU-S), and B6-W41J/W41J mice are fertile. Nevertheless, lymphoid and erythroid repopulation occur to a similar extent in B6-W41J/W41J or -W41J/W39J and in WBB6F1-W/Wv anemic mutants. Repopulation is more rapid in the latter, but host cells may be damaged by B6 reactions against the WB parent. Avoiding graft-versus-host reactions, hybrid resistance, and similar complications are important advantages in using donors and unirradiated recipients all on the B6 mouse genetic background. Additionally, congenic B6 mice provide a variety of genetic markers, allowing myeloid and lymphoid repopulation to be readily quantitated.

Effects of transplantation and age on immunohemopoietic cell growth in the splenic microenvironment.

Intact spleens from young adult and aged mice were transplanted into young recipients to compare effects of age and effects of spleen transplantation on hemopoietic and immune functions. Hemopoietic functions of histocompatible spleen transplants were assessed by partial cures of genetically anemic WBB6F1-Sl/Sld recipients, and immune functions were measured as numbers of anti-SRBC PFC(sheep red blood cell plaque-forming cells) and responses to the mitogen PHA (phytohemagglutinin). Spleens from WCB6F1 and WBB6F1 donors at least 28 months old partially corrected anemias in 10 of 28 Sl/Sld recipients, whereas spleens from 5- to 10-month-old donors performed significantly better, partially correcting anemias in 22 of 31 Sl/Sld recipients. B6D2F1 spleens were transplanted from either old or young donors in B6D2F1 recipients to test their ability to support immune-responsive cells. These spleen grafts were much smaller than recipient spleens and contained few anti-SRBC PFC. In contrast WCB6F1-+/+ spleens transplanted in Sl/Sld recipients were much larger, weighing more than the intact spleens of the recipients. Nevertheless when these spleens were from young donors, they contained only about 10% as many anti-SRBC PFC and PHA-responsive cells as did recipient spleens, whereas old donor spleens contained even fewer. Use of splenectomized Sl/Sld recipients did not alter these results. Apparently the effect of transplantation was much more important than age in reducing the spleens' abilities to support immune-responsive cells.

Development and aging of primitive hematopoietic stem cells in BALB/cBy mice.

Evaluating the function of an individual hematopoietic stem cell (HSC) is a difficult and important problem. The functional ability per HSC, as well as the HSC concentration, was measured with minimal disruption to the cells in vivo using the new competitive dilution assay. Distribution of HSC into recipients was modeled based on Poisson probabilities. Predictions of donor contributions from models assuming different levels of donor HSC functional ability and concentration were compared to actual observations. The model with the least difference between predictions and observations was accepted. In BALB/ cBy (BALB) mice, models assuming equal functional ability of HSC from the same donor fit extremely well with actual observations, suggesting that all HSC are functionally homogeneous at any particular time point during development or aging. Relative HSC functional ability per cell declined during development, so that a fetal HSC had 1.6 to 3.0 times the functional ability of a young adult HSC. The decline continued with age, so that a young adult HSC had 1.6 to 2.0 times the functional ability of an old HSC. Concentrations of HSC that engrafted and functioned were similar among 16-day fetal liver cells and bone marrow cells (BMC) from 3-month and 25 to 28-month-old adult mice. They were either 10 or 4 HSC per million cells when tested in BALB or CByB6F1 recipients, respectively. All HSC were pluripotent and produced lymphoid and myeloid descendants proportionally (r = 0.80 to 0.98, p < 0.01). Fetal and young HSC in both types of recipients maintained clonal stability long term so that percentages of donor cells at 6 and 9 months were strongly correlated (r = 0.72 to 0.93, p < 0.01). Although HSC from aged donors in BALB recipients maintained clonal stability, HSC from the same aged donors failed to show clonal stability in CByB6F1 recipients, perhaps due to the less suitable host environment. All HSC from BALB mice seemed to have equal functional levels at a given stage of life and were gradually exhausted simultaneously through development and aging.

Genetic regulation of primitive hematopoietic stem cell senescence.

OBJECTIVE: To define effects of strain on PHSC (primitive hematopoietic stem cells) senescence (decline in function with age) in vivo, and to map a locus that regulates PHSC senescence. MATERIALS AND METHODS: Long-term function and self-renewal were compared in bone marrow cells (BMC) from old and young mice of three strains: BALB/cBy (BALB), DBA/2 (D2) and C57BL/6 (B6), using competitive repopulation and serial transplantation in vivo. BMC from each old or young donor were mixed with standard doses of congenic, genetically marked BMC and transplanted into lethally recipients. Percentages of donor-type erythrocytes and lymphocytes in the recipients determined the functional ability of donor PHSC relative to the standard, where one repopulating unit (RU) of donor BMC equals the repopulating ability of 100,000 standard competitor BMC. Using similar techniques, repopulating abilities of old and young recombinant inbred (RI) donors of 12 strains derived from BALB and B6 were compared in NK-depleted BALBxB6 Fl recipients to map a locus that appears to have a major role in PHSC senescence. RESULTS: PHSC function declined about 2 fold with age in BALB and D2 BMC, and increased more than 2-fold with age in B6 BMC, with all old/young strain differences significant, p<.01. Ten months after serial transplantation, young B6, BALB, and D2 PHSC had self-renewed 1.6-, 4.2-, and 3.2-fold better than old, with BALB and D2 old/young differences p<.01. Young B6 PHSC self-renewed 1.9- and 2.9-fold better than young BALB and D2 PHSC. The PHSC senescence phenotypes (old/young RU ratios) for 12 CXB RI strains suggested a genetic linkage to D12Nyul7 on Chromosome 12. CONCLUSION: PHSC senescence is genetically regulated, and is much delayed in the B6 strain compared to the BALB and D2 strains. A locus on Chromosome 12 may regulate PHSC senescence.

Hematopoietic precursor cell exhaustion is a cause of proliferative defect in primitive hematopoietic stem cells (PHSC) after chemotherapy.

The authors used the competitive repopulation assay and simple statistical analyses to estimate concentrations of primitive hematopoietic stem cells (PHSCs) in the marrow of mice after chemotherapy. Single doses of cyclophosphamide (CTX) from 80 to 200 mg/kg were administered to C57B16/J mice. Other treatment groups included mice given multiple doses of CTX at the lowest dose of 80 mg/kg; mice given four weekly doses of vincristine (VCR) or vinblastine (VBL); mice given two biweekly doses of bleomycin; mice receiving cytosine arabinoside (ARA) administered intraperitoneally thrice daily or as a continuous infusion by Alzet pump for 3 days; and controls given no drug. The lowest dose of CTX (80 mg/kg), given once or repeatedly, spared PHSC numbers and function. The functional capacity of PHSCs declined significantly once doses of CTX exceeded 100 mg/kg. Decreases in PHSC function were usually associated with reductions in PHSC numbers; repopulating units, which include all repopulating cells, were similarly reduced. At the highest dose (33 mg/kg for 3 days), ARA caused a decline in marrow repopulating function. Drugs associated with mild clinical myelosuppression, such as VCR and VBI, did not significantly affect the repopulating ability of PHSCs, although VCR caused drastic declines in PHSC numbers. The marrow reconstitutive defects clinically-observed after chemotherapy may be caused partly by depletion of the PHSC pool.

The decrease in long-term marrow repopulating capacity seen after transplantation is not the result of irradiation-induced stromal injury.

Marrow cells from nonirradiated F1-W/Wv mice repopulated slightly less well than cells from lethally irradiated recipients. Therefore, avoiding irradiation of recipients did not improve the relative repopulating ability of their marrow cells. In other experiments, F1-W/Wv mice were transplanted by parabiosis with marrow of WBB6F1-+/+ (F1-+/+) mice, avoiding cellular handling and irradiation. Marrow cells transplanted to F1-W/Wv mice by this procedure demonstrated slightly better repopulating ability than did marrow cells transplanted by injection. However, they performed no better than those transplanted by parabiosis to irradiated F1-+/+ recipients. Significant impairment of stromal function after irradiation was not indicated. Apparently, stem cell damage caused by transplantation may have greater importance in causing loss of stem cell replicative potential than effects of irradiation-induced stromal injury.

Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term.

Multilineage precursor cells from 14-day B6 (C57B1/6J) mouse fetal liver and adult bone marrow that repopulate both the lymphoid and myeloid systems were compared by competitive repopulation. Cells were assayed in normally functioning populations, and enrichment, tissue culture, and induced marking were avoided since these manipulations might affect cell function. Fetal or adult donor cells were mixed with marked adult competitor cells and transplanted into irradiated recipients whose blood was tested at short (25-33-day) or long (105-245-day) time periods after transplantation. Proportions of lymphocytes, granulocytes, and platelets descended from donor precursors were measured by GPI (glucosephosphate isomerase) isozyme genetic markers in congenic mice, and represent the repopulating abilities of these precursors relative to the standard competitor. For short-term repopulation 25-33 days after transplantation, fetal and adult donor cells were similar; in three studies, fetal liver contributed 0.8, 1.1, and 1.4 times as much as adult marrow per 10(5) cells transplanted. However, when long-term (105-245-day) repopulation was tested in the same recipients, fetal liver contributed 3.5, 5.0, and 7.1 times as much as adult marrow. Ratios of long-term/short-term repopulating abilities in fetal liver relative to standard adult marrow competitors were 2.5, 8.9, and 4.7, while in marrow controls, these ratios remained approximately one (1.14 and 0.80). Thus, 14-day fetal liver contains several times more long-term repopulating cells relative to short-term repopulating cells than does adult marrow. Ratios of long-term/short-term fetal cells were unchanged by precursor enrichment. The AA4.1+, Ly-6A/E+, lineage low fraction had a ratio of 4.4, although it repopulated 276 times better than unenriched fetal cells whose ratio was 4.7. There are two hypotheses that explain these data most simply: 1) There may be only a single multilineage precursor, but after transplantation cells seed in different microenvironments that support either long-term or short-term function. 2) Conversely, the difference may be at the stem cell level rather than the microenvironmental level, so that there are tow types of stem cells with multilineage differentiating ability, but only one functions over the long-term. The current report defines new conditions required by each hypothesis. If functional life spans are defined by seeding sites, as in hypothesis 1, fetal cells seed much higher proportions of long-term sites than adult cells. If different types of stem cells function short-term and long-term, as in hypothesis 2, they are not distinguished by markers allowing a 276-fold enrichment to 1367 times the repopulating ability of fresh marrow.

Long-term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation.

To characterize hematopoietic cell biology, many investigators have used protocols that enrich for primitive hematopoietic stem cells (PHSC). In this study, we quantified the long-term repopulating ability (LTRA) of enriched and discarded fractions of PHSC from day-14 murine fetal liver using the competitive repopulation assay. We fractionated populations of fetal cells using the antigenic markers AA4.1+, AA4.1+/Sca+, and AA4.1+/Linlow/Sca+. Differentiating and repopulating abilities of each of these populations were directly compared using competitive repopulation. Adult bone marrow was mixed with fetal cell fractions from congenic donors having genetically distinguishable markers, and mixtures were given to irradiated recipients. Differentiating and repopulating abilities of the enriched donor cells were measured by the proportions of myeloid and lymphoid cells having donor markers that repopulated the recipients. LTRA was found primarily in the AA4.1+ and AA4.1+/Sca+ subpopulations. Further fractionation of the AA4.1+ cells to derive an AA4.1+/Linlow/Sca+ fraction showed that virtually all of the long-term stem cell activity was found in this subpopulation. These cells were 1400- to 1600-fold enriched in long-term functional ability compared to fresh marrow. This very high multilineage repopulating ability per cell was directly measured using a long-term functional assay in vivo. Importantly, the measured repopulating ability for AA4.1+/Linlow/Sca+ cells was about five-fold less than expected from the fraction of cells enriched and remained two- to three-fold less even after compensating for repopulating ability in discarded fractions. This illustrates that long-term functional abilities of enriched PHSC cannot be estimated from fractions enriched but should be quantitatively assayed.

Primitive hemopoietic stem cells: direct assay of most productive populations by competitive repopulation with simple binomial, correlation and covariance calculations.

Quantitative analyses of primitive hemopoietic stem cell (PHSC) populations are important both for basic biology and for clinical applications. Unfortunately, many conventional assays fail to measure long-term repopulating ability and maximal differentiating ability, the most important characteristics of the PHSC. The competitive repopulation assay described here focuses on this characteristic, assaying the precursors from which most differentiated cells are descended over large fractions of the life span in laboratory mice. Thus long-term repopulating ability and the ability to differentiate into both myeloid and lymphoid lineages are measured directly from 2.5 to 12.5 months after transplantation. This technique also has found high correlations between granulocytes, macrophages, and T and B lymphocytes as early as 3 weeks after transplantation. All or most differentiated cells of these widely disparate types appear to be descended from a common precursor cell, while myeloid-specific or lymphoid-specific precursors produce few or no descendants. However, large increases in variances between 3 and 6 weeks and 12 weeks after transplantation suggest that most of the initially active multilineage precursors are exhausted. Thus the ability to differentiate into widely disparate lineages does not establish long-term repopulating ability.

Graphenylene Nanotubes.

A new type of carbon nanotube, based on the graphenylene motif, is investigated using density functional and tight-binding methods. Analogous to conventional graphene-based nanotubes, a two-dimensional graphenylene sheet can be "rolled" into a seamless cylinder in armchair, zigzag, or chiral orientations. The resulting nanotube can be described using the familiar (n,m) nomenclature and possesses 4-, 6-, and 12-membered rings, with three distinct bond lengths, indicating a nonuniform distribution of the electron density. The dodecagonal rings form pores, 3.3 Å in diameter in graphenylene, which become saddle-shaped paraboloids in smaller-diameter nanotubes. Density functional theory predicts zigzag nanotubes to be small-band gap semiconductors, with a generally decreasing band gap as the diameter increases. Interestingly, the calculations predict metallic characteristics for armchair nanotubes with small diameters (<2 nm), and small-band gap semiconducting characteristics for larger-diameter ones. Graphenylene nanotubes with indices mod(n-m,3) = 0 exhibit a band gap approximately equal to that of armchair graphenylene nanotubes with comparable diameter.