Isolation of murine pluripotent hemopoietic stem cells.

A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation.

Photooxidation of chlorophyll in spinach chloroplasts between 10 and 180 K.

Electron paramagnetic resonance (EPR) and optical absorbance difference spectra and kinetics upon illumination by saturating flashes and continuous light of spinach chloroplasts frozen under various conditions were measured between 10 and 180 K. 1. At 100 K illumination with continuous light caused an EPR signal which decayed during the light in about 30 ms. This change is probably due to the reduction of P+-680, the oxidized primary electron donor of Photosystem II, by a secondary electron donor, cytochrome b-559. Flash illumination yielded the previously observed rapid (2 ms) transient. This transient has been ascribed to a back-reaction of the two primary reagents of Photosystem II (Malkin, R. and Bearden, A.J. (1975) Biochim. Biophys. Acta 396, 250-259; Visser, J.W.M. (1975) Thesis, Leiden). 2. Between 10 and 40 K, illumination with continuous light showed a transient which decayed in about 500 ms. The extent decreased with increasing temperature. However, the half time appeared to be temperature independent. This signal was also attributed to P+-680. 3. At 180 K it appeared to be impossible to observe the 2 and 30 ms components in dark frozen chloroplasts. However, they could be observed again if two short saturating flashes were given shortly before freezing. These changes seem to be dependent on the S-state of the reaction center. 4. After oxidizing the sample with ferricyanide (Eh = 540 mV), the light induced absorbance difference spectrum showed a bleaching near 676 nm. This change is ascribed to the irreversible oxidation of a dimeric chlorophyll molecule which acts as electron donor to P+-680 under these conditions. 5. Titration curves of the irreversible light-induced absorbance change at 676 nm and the irreversible light-induced EPR change near g = 2.00 provide strong evidence that these two changes reflect the same compound. Finally, a model is given to explain the observed reactions of Photosystem II at 10-180 K. The model involves three different ultimate and one intermediate electron donor to P+-680 at these temperatures.

EPR signals of oxidized plastocyanin in intact algae.

(1) An electron paramagnetic resonance (EPR) signal was observed at g = 2.05 in the low temperature spectra of intact cells of green, red and blue-green algae and of spinach chloroplasts. The g-value and the shape of the signal were similar to that of purified, soluble plastocyanin. (2) The amount of the copper protein, determined from the EPR signal height, was estimated to be nearly the same in all the studied organisms on the basis of the concentration of chlorophyll. Furthermore, it was found that the amount of the copper protein, determined from the EPR signal height in spinach chloroplasts corresponds with that of plastocyanin as determined chemically by Katoh, S., Suga, I., Shiratori, I. and Takamiya, A. (1961) Arch. Biochem. Biophys. 94, 136-141. (3) Experiments with far-red and red illumination show that the site of the copper protein in vivo is in the electron transport pathway between Photosystems 1 and 2. Plastocyanin is not oxidized by illumination at 77 degrees K, indicating that no electron transfer occurs between the primary electron donor of Photosystem 1, P700, and plastocyanin at that temperature. Furthermore, the experiments suggest that in the intact cells of the studied algae, plastocyanin is not only reduced by Photosystem 2 but also by cyclic electron transport around Photosystem 1.

Purification of repopulating hemopoietic cells based on binding of biotinylated Kit ligand.

To characterize Kit expressing mouse bone marrow (BM) cells, and to determine their contribution to short- and long-term repopulation of the hemopoietic system of irradiated recipients, we have purified Kit+ BM cells by flow cytometry. A high level of Kit expression was detectable on 1-2% of BM cells after staining with biologically active biotinylated Kit ligand (KL) or with anti-Kit antibodies (ACK-2). Compared to unfractionated BM, the Kit+ fractions were enriched for immature hemopoietic cells, as shown by morphological differentiation, in vitro culture, and spleen colony formation. Enrichment of colony-forming cells was higher in biotin-KL+ than ACK-2+ fractions. Colony-forming cells were not found in the Kit- subsets. To study the hemopoietic repopulation capacity of the Kit+ and Kit- cells, serial dilutions of the sorted fractions were transplanted into irradiated mice, and peripheral blood of these recipients was monitored regularly for the presence of donor-derived cells during a 1 year period. Nucleated blood cell repopulation by male donor cells in female recipients was assessed using a Y-chromosome specific DNA probe; erythroid repopulation by normal donor cells in W/Wv recipients was examined flow cytometrically by measuring the forward light scatter of donor- and host-type erythrocytes. A 25- to 100-fold enrichment of long-term repopulating ability in the sorted Kit+ fractions showed that Kit+ cells are capable of reconstitution of circulating erythrocytes and nucleated blood cells after BM transplantation. Transient repopulation of the red blood cell lineage was observed after transplantation of Kit- cells. Detection of donor-derived nucleated cells 1 year after transplantation showed that Kit+ cells contributed to donor-type repopulation of bone marrow, spleen and thymus. Our data demonstrate that isolation of BM cells on the basis of Kit expression is a useful addition to the methods that are commonly applied in stem cell enrichment protocols.

Monitoring of leukemia growth in a rat model using a highly sensitive assay for the detection of LacZ marked leukemic cells.

A very sensitive assay for the detection of LacZ marked cells of an in vitro growing subline of the brown Norway rat myelocytic leukemia (BNML) model was developed. By combining cytochemical X-gal staining with D-galactose mediated suppression of endogenous background beta-galactose activity, a detection sensitivity of one leukemic cell per 10(8) normal bone marrow cells could be achieved. A detailed analysis of the in vivo growth pattern and kinetics of this cell line is presented. Also, it is shown that after cyclophosphamide treatment of leukemic rats no leukemic colonies are formed in an agar-colony assay, whereas the leukemic cells remain detectable in the bone marrow for a considerable time period. Eventually, however, all leukemic cells disappear from the marrow. These findings are discussed in the light of prolonged detection of rare leukemic cells in patients in continuing remission.

Separation and functional analysis of bone marrow cells separated by rhodamine-123 fluorescence.

Mouse bone marrow (BM) cells were separated on the basis of fluorescence intensity after labeling with the supravital, fluorescing dye rhodamine 123 (Rh 123). Rh 123 accumulates in the mitochondria. The BM fractions were tested for the presence of spleen colony-forming units (CFU-S) that produced colonies at day 8 and day 12 after bone marrow transplantation; for the ability to rescue lethally irradiated mice; and for thymus-repopulating ability. The results showed that all the day-8 CFU-S incorporated a relatively large amount of Rh 123, while the day-12 CFU-S were stained heterogeneously. Survival after lethal irradiation, as expressed in the numbers of day-12 CFU-S transplanted, was predominately mediated by the weakly fluorescing fraction. However, early thymus repopulation, which is caused by prothymocytes contained in the graft, was mediated by the brightly fluorescing fraction. Data in the literature indicate that a high uptake of RH123 is correlated with cellular proliferation. This suggests that all the day-8 CFU-S and about 60% of the day-12 CFU-S are cycling. In contrast, less than 10% of both day-8 and day-12 CFU-S are killed by S-phase-specific agents. From this we conclude that a high uptake of Rh 123 depends on other factors in addition to the cell cycle status. It is suggested that differentiation processes in which the day-8 CFU-S appears to be involved also cause the presence of many or very active mitochondria. The difference between the weakly and brightly fluorescing fractions in the number of day-12 CFU-S required for 30-day survival after lethal irradiation, suggests that there is heterogeneity among the day-12 CFU-S population or that the fraction weakly labeled with Rh123 contains other, less mature, cells that are responsible for survival after lethal irradiation. Finally, the presence of prothymocytes in the brightly labeled fraction shows that these cells are different from the stem cells that protect lethally irradiated mice.

Proliferation of purified murine hemopoietic stem cells in serum-free cultures stimulated with purified stem-cell-activating factor.

Under conditions of steady-state hemopoiesis in normal mice, the majority of hemopoietic stem cells in the bone marrow are in the quiescent state of the cell cycle. These cells can be stimulated to proliferate in vitro by the addition of a factor termed the "stem-cell-activating factor" (SAF), which is present in medium conditioned by various cell types. This factor is indistinguishable in antigenic and molecular properties from the lymphokine interleukin 3 (IL-3). The action of SAF on the stem cell cycle was studied by examination of the survival of the spleen colony-forming unit(s) (CFU-S) after four days of serum-free culture in the presence of purified SAF. CFU-S subtypes were distinguished on the basis of the day of colony counting (i.e., days 7, 9, and 12 after transplantation). The results indicate that SAF selectively induces an increase of the day-7 CFU-S: the CFU-S number increased 2.7-fold on day 7 and 1.2-fold on day 9, and decreased fivefold for day-12 CFU-S. Similar results were obtained in SAF culture of partially and highly purified stem cells. The proliferation of day-9 CFU-S in cultures of low-density bone marrow cells was found to be similar to that of unfractionated bone marrow cells until day 4 of culture. However, in the culture of partially purified stem cells, this proliferation stopped between days 4 and 10, whereas it continued with unfractionated cells. This indicates that cocultured bone marrow cells affect the proliferation of stem cells upon induction by SAF; day-4 cultures of highly purified resting stem cells with purified SAF resulted in a similar decrease in day-12 CFU-S and an increase in day-7 CFU-S, as was observed with unfractionated bone marrow cells. The DNA histogram of the stimulated sorted cells clearly revealed an actively DNA-synthesizing population. The results are in agreement with those of a selective induction of proliferation by SAF of resting pluripotent hemopoietic stem cells.

Analysis and separation of murine bone marrow stem cells by H33342 fluorescence-activated cell sorting.

Murine bone marrow cells were stained with the fluorescent bisbenzimide dye H33342, a supravital DNA stain, and sorted on the basis of differences in fluorescence intensity by a light-activated cell sorter. Sorted cells were submitted to assays to enumerate spleen colony forming cells (CFUs), HPP-CFC and GM-CFU-1 and -2. The recoveries of these cell types after the separation was 70 to 120%, except for GM-CFU-2 (10-40%). The frequency distribution of these cell types with respect to their fluorescence intensity suggested that the majority of CFUs and HPP-CFC are quiescent, whereas GM-CFU-2 are proliferating. Furthermore, HPP-CFC could be separated from GM-CFU-1 and -2 on the basis of fluorescence intensity differences, which indicates that these cell types are different. CFUs determined 10 days after irradiation and grafting of the recipient mice were distributed evenly over the two fluorescence intensity subpopulations which contained most of the HPP-CFC and the GM-CFU. The same was observed at day 8. However, CFUs determined at day 12 were only present in the subpopulation of low fluorescence intensity which also contained most of the HPP-CFC. This observation provides evidence for heterogeneity of the spleen colony forming cells.

Thymus regeneration by bone marrow cell suspensions differing in the potential to form early and late spleen colonies.

We have determined the thymus-repopulating capacity of purified hemopoietic stem cells, bone marrow cells from mice injected four days previously with 5-fluorouracil (5-FUBM), and bone marrow cells cultured in the presence of stem-cell-activating factor (SAF; SAFBM). SAF is identical to interleukin 3 (IL-3). Purified stem cells are more enriched in day-12 CFU-S than in day-8 CFU-S. 5-FUBM consists of CFU-S that give rise to late (day-12) spleen colonies. SAFBM contains predominantly CFU-S that give rise to early spleen colonies (days 6-8). There is also a net increase in the number of spleen colony-forming units (CFU-S) in these cultures. Thymus regeneration after transplantation with either purified stem cells or 5-FUBM was delayed approximately three days as compared with that after transplantation with normal bone marrow cells. This delay can be ascribed to the absence of prothymocytes in these preparations. Thymus regeneration by SAFBM was delayed approximately ten days as compared with that after transplantation with normal bone marrow cells. The most likely explanation of these results is as follows. The prothymocytes in normal bone marrow produce a relatively limited offspring in the thymus soon after transplantation. This is rapidly replaced by the offspring of newly formed prothymocytes, the results of differentiation of the pluripotent stem cells. These stem cells also give rise to late spleen colonies. Stem cells that give rise to early spleen colonies appear to have lost the capacity for differentiation into the T-cell lineage.

Characterization and enrichment of murine hemopoietic stem cells by fluorescence activated cell sorting.

Several methods for supravital staining of mouse bone marrow cells are described. These methods were employed to sort fractions of bone marrow cells by a light activated cell sorter and to determine cytochemical properties of hemopoietic stem cells. Labelling with fluoresceinated wheat germ lectin confirmed that the stem cells are among the bone marrow cells with the highest surface density of N-acetylneuraminic acid. Staining with tetracycline revealed that the stem cells contain relatively active or relatively many mitochondria. Staining with the bisbenzimide H33342, a supravital DNA stain, indicated that most pluripotent stem cells are in a G0/G1 state of the cell cycle. Using these and other measurements, separation protocols can be devised to concentrate stem cells. Some of these are discussed.

Analysis of gene expression in subpopulations of murine hematopoietic stem and progenitor cells.

OBJECTIVE: The aim of the present work was to study how functional differences between subsets of the murine hematopoietic stem/progenitor cell compartment are manifested on the level of different patterns of gene expression in these subsets. MATERIALS AND METHODS: Amplified 3' terminal total cDNA fragment populations from four stem and progenitor cell fractions sorted using differential staining with Rhodamine 123 were prepared, and gene expression patterns were analyzed by Southern hybridization with a panel of gene markers. RESULTS: For the vast majority of lineage-specific markers, no expression was detected in the long-term repopulating stem cell fraction. Expression of a number of key genes positively regulating entry and progression through the cell cycle was down-regulated in long-term repopulating cells, in accordance with the quiescent state of the latter. In contrast, certain but not all cell division kinase inhibitors were significantly up-regulated in long- and short-term repopulating stem cell fractions. Expression of several genes important for entry into the apoptotic pathway was moderately reduced in long-term repopulating cells. Messenger RNA levels of the transcription factors GATA-1, GATA-2, c-Myb and SCL were down-regulated in long-term repopulating cells, as compared to more mature stem/progenitor cells. Finally, expression of the MDR1a gene encoding the Pgp efflux pump was highest in long-term repopulating cells, and progressively decreased with maturation. CONCLUSION: The patterns of gene expression in the stem/progenitor cell fractions are in good correlation with the known properties of adult hematopoietic stem/progenitor cells and may provide insight into molecular mechanisms underlying stem cell physiology.

Homing of fluorescently labeled murine hematopoietic stem cells.

PKH-26 was used as a viable fluorescent membrane stain for murine hematopoietic stem cells. The presence of the dye on the cells was shown not to interfere with their ability to form day-8 and -12 spleen colonies in lethally irradiated mice. To study their in vivo homing behavior in detail, 10(4) labeled cells from a population enriched for CFU-S were injected intravenously into nonirradiated mice and into mice irradiated 3 hours previously. At 17, 41, and 65 hours after injection, the numbers of labeled cells per organ were quantified using the specialty developed flow cytometric fluorescence hypercompensation procedure for the detection of rare events, which allows a detection sensitivity of 1 per 10(6). Spleen homing in irradiated and nonirradiated mice was virtually identical, whereas homing to nonirradiated bone marrow was 2.5 times higher than to irradiated bone marrow. This indicates a different homing mechanism for spleen and bone marrow. The results of this direct homing assay were placed in perspective with results of indirect homing studies from the literature, introducing a new "h-factor." From the CFU-S data, putative specific enrichment factors for spleen-specific and bone marrow-specific homing were derived. Examination of the fluorescence intensity distribution among the labeled cell population indicated that virtually all cells started to proliferate rapidly after injection into both irradiated and nonirradiated animals. This indicates that specific signals from stromal elements in the stem cell niches are needed to keep the cells quiescent and that the majority of the transplanted stem cells do not home to such niches. The potential use of PKH-26 for in vivo characterization of stem cell niches is discussed.

Culture of hematopoietic stem cells purified from murine bone marrow.

The results of the Y-chromosome in situ hybridization experiments, the MRA assessment, and the long-term production of CFU-GM in vitro indicate that our protocol to sort low density WGA+, 15/1.1-, Rh123 dull cells enriches about 200-fold for PHSC. Assays for spleen colony formation (CFU-S) and radioprotection (30-day survival) were shown to be unspecific for PHSC, and, therefore, we lack a quantitative PHSC assay. The absolute number of PHSC in the bone marrow is not known any more, the purity of our sorted population likewise is unknown. Long-term repopulating cells (PHSC) could be separated from short-term repopulating ones by using Rh123 staining. The short-term repopulating cells (Rh123 bright) provided sufficient offspring to protect lethally irradiated mice until endogenous PHSC could reconstitute hematopoiesis. These cells are therefore of interest for bone marrow transplantation, because they provide radioprotection without long-term repopulation and graft-versus-host disease. For gene therapy these cells are of limited use, and PHSC with extensive replication are needed. The PHSC were not cultured successfully. Less than 15% of the sorted Rh123 dull cells responded in semisolid or liquid cultures in the presence of growth factors. Proliferation without differentiation was not observed. This may indicate that the right growth factor has not been found yet. On the other hand, about 30% of the cells responded in stromal layers of long-term bone marrow cultures and prolonged CFU-GM production and cobblestone area formation were observed there, suggesting that cell-cell contact and adherence molecules play a regulatory role in PHSC replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel CDC2-related protein kinases produced in murine hematopoietic stem cells.

The polymerase chain reaction with degenerate primers was used for the amplification of cDNA encoding CDC2-related protein kinase (PK) sequences from murine hematopoietic stem cells. In total, nine different PK-encoding sequences were obtained. At least four of them encode previously unknown PKs.

Genomic structure and alternative splicing of the murine bhk/ctk/ntk gene.

Recently, we and others have cloned cDNAs encoding a second member of the Csk family of inhibitory protein kinases, which we termed Bhk [M.A. Ershler et al. (1994) Dokl. Akad. Nauk. 339, 679-683]. In the present study, two new distinct types of bhk mRNA were found in addition to the third form described previously. Analysis of the bhk genomic structure established that three exons participate in the alternative splicing of bhk mRNA.

A novel murine cathelin-like protein expressed in bone marrow.

A novel cDNA encoding a putative secreted protein was isolated from murine bone marrow. The encoded protein named MCLP (murine cathelin-like protein) was found to be highly homologous to the pig cathelin, and to four neutrophil antimicrobial polypeptides: CAP 18, indolicidin, Bac 5 and FALL-39. Secondary structure prediction studies identified a highly cationic region in the C-terminal part of prepro-MCLP with a tendency to adopt an amphipathic alpha-helical conformation, as observed in many antimicrobial peptides. However, no antibacterial activity was observed with the synthetic peptide corresponding to this region of MCLP.

Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods.

The main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that binding of the biotinylated ligand to the receptor does not inhibit the subsequent interaction of biotin with fluorescently tagged avidin or streptavidin. Using interleukin-2 (IL-2), we compared the usefulness of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS-LC-biotin, DBB and photobiotin, and developed optimal biotinylation conditions for the preparation of biologically active biotin-labeled IL-2 and the detection of IL-2 receptor expressing cells by flow cytometry. As determined by spot blot analysis, biotinylation of IL-2 was most efficient at the highest biotin-to-protein (B:P) ratio used. At a B:P ratio of 100, most of the biological activity of IL-2 was retained when S-NHS-LC-biotin was used. In contrast, most of the biological activity of IL-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions. Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cells by flow cytometry after sequential staining with the biotinylated IL-2 and fluorescence tagged streptavidin. A high B:P ratio generally resulted in a high specific fluorescence intensity of the cells, particularly when S-NHS-LC-biotin was used as the biotinylation reagent. Biotin-IL-2 could also be used to detect IL-2 receptors expressed by lymphocytes in peripheral blood and bone marrow. Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 receptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated IL-2 expressed high numbers of the IL-2 receptor alpha chain. This is in agreement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain. After stimulation with PHA, virtually all lymphocytes expressed the alpha chain, whereas only part of these cells showed a strong fluorescence signal after staining with biotin-IL-2, while the rest of the cells had very low numbers of IL-2 binding sites. Our results demonstrate that, in addition to staining individual receptor subunits with antibodies, staining with biotinylated IL-2 is a useful indicator of functional IL-2 receptor expression.

Bone marrow transplantation in newborn rats with mucopolysaccharidosis type VI: biochemical, pathological, and clinical findings.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB). In this study, we evaluated bone marrow transplantation (BMT) for the treatment of MPS VI and the effects of irradiation on the survival and engraftment of bone marrow-transplanted neonatal rats. METHODS: One- to 2-day-old MPS VI rats were injected with normal bone marrow after irradiation with 200, 400, or 800 cGy. Ninety percent of the animals receiving a single dose of 200 cGy (n=30) survived the procedure, whereas irradiation with 400 cGy (n=23) or 800 cGy (n=12) resulted in significant mortality (78% and 100%, respectively). Engraftment was monitored by determining ASB activities in peripheral white blood cells and by Y chromosome in situ hybridization analysis. Fifty-two percent of the animals from the 200-cGy group engrafted for up to 8 months after BMT; among the five animals that survived the 400-cGy dose, all engrafted. In comparison, only 20% of nonirradiated animals engrafted at low levels. Of the 24 engrafted animals that were monitored for 8 months after BMT, clinical and/or radiographic improvements were noted in only one (BMT animal 3). Enzymatic analysis revealed that the ASB activities in the reticuloendothelial organs of this animal, as well as two other engrafted but clinically unimproved animals (BMT animals 1 and 2), were normal or near normal; correspondingly, the glycosaminoglycan levels in these organs were significantly reduced. Consistent with the clinical and biochemical observations, light and electron microscopic findings were more improved in BMT animal 3 as compared with BMT animals 1 and 2, although a reduction of storage was evident in each of these transplant recipients, particularly in the trachea and aorta, two tissues that are characteristic sites of pathology in human patients. CONCLUSIONS: These results indicate that BMT in newborn MPS VI patients may prevent many of the pathological and clinical findings in this disorder, but is likely to have very limited and unpredictable effects on the skeletal abnormalities.

Intracellular pH-determination by fluorescence measurements.

A method was developed to determine the intracellular pH (pHi) of individual cells by use of fluorescence measurements. The method is based on the observation that the fluorescence excitation spectrum of fluorescein is pH-dependent. Fluorescence excitation spectra from individual rat bone marrow cells treated with fluorescein diacetate (FDA) were compared with those of fluorescein solutions of known pH values. Cells which were suspended in media of pH between 4.0 and 8.1 with high to normal buffering capacities had pHi values equal to those of the media. Cells suspended in media with low buffering capacities maintained a pH,i of 6.7 +/- 0.2. Preliminary results indicated that the pHi of individual cells may also be determined by using flow cytometry.

CD3+, 4+ and/or 8+ T cells and CD3+, 4-, 8- T cells repopulate at different rates after allogeneic bone marrow transplantation.

The antigen receptors of the majority of peripheral blood T lymphocytes are constituted of alpha- and beta-chains in association with CD3. The phenotype of those T cell receptor-alpha, beta cells is CD3+, 4+ and/or 8+. The small subset of CD3+, 4-, 8- T cells includes TCR-gamma, delta cells. These two T cell subsets have different TCR gene rearrangement patterns, tissue distributions and mechanisms of antigen recognition. We studied the repopulation of both T cell subsets in 20 allogeneic marrow graft recipients in relation to the type of graft (T cell-depleted versus non-depleted) and the occurrence of active cytomegalovirus (CMV) infection, using three-color immunofluorescence and flow cytometry. The CD3+, 4+ and/or 8+ and CD3+, 4-, 8- T cells had clearly different repopulation patterns. At 1 month post-BMT, they had repopulated the blood to similar levels. Thereafter, the CD3+, 4+ and/or 8+ T cells increased further in number, whereas the CD3+, 4-, 8- T cells stabilized on average between 100 and 200 x 10(6)/l. The nine recipients of T cell-depleted marrow grafts showed a relatively delayed repopulation of their CD3+, 4+ and/or 8+ T cells compared with the 11 recipients of non-depleted marrow. In contrast, the repopulation rate of the CD3+, 4-, 8- T cells was similar in both groups. The occurrence of active CMV infection post-BMT was associated with an increased rate of repopulation of the CD3+, 4+ and/or 8+ T cells, particularly those expressing HNK1, but did not affect the repopulation of the CD3+, 4-, 8- T cells.