Characterization and phenotypic analysis of differentiating CD34+human bone marrow cells in liquid culture

Abstract: Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34⁺ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34⁺ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34⁺ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.     

Neonatal exposure to estradiol prevents the expression of ovarian hormone-induced luteinizing hormone and prolactin surges in adulthood but not antecedent changes in neuropeptide Y or adrenergic transmitter activity: Implications for sexual differentiation of gonadotropin secretion

Sex differences in adult patterns of mating behavior and gonadotropin secretion in rats are determined in part by the presence or absence of gonadal steroids during a perinatal critical period. For example, male rats and female rats exposed neonatally to androgen do not exhibit LH surge patterns when treated appropriately with ovarian hormones in adulthood, and there is evidence that this may be due to a failure of ovarian hormones to activate the hypothalamic neuronal systems that stimulate LH secretion in such animals. Because considerable evidence suggests that estradiol formed centrally from testosterone is responsible for the permanent defeminization of mating behavior and gonadotropin secretion, the present studies compared normal females with normal males and with females treated neonatally with estradiol on the ability of ovarian hormones to induce several important neurochemical changes antecedent to the LH surge, including changes in neuropeptide Y (NPY) and LH-releasing hormone (LHRH) concentrations in the median eminence, as well as changes in turnover rates for catecholamine transmitters in the medial basal hypothalamus and medial preoptic area. Normal ovariectomized female rats responded to sequential treatment with estradiol followed by progesterone with afternoon LH and prolactin (PRL) surges, and with sequential accumulation followed by decline in concentrations of LHRH and NPY in the median eminence prior to the LH surge. In addition, administration of progesterone increased the turnover rates of norepinephrine (NE) and epinephrine (EPI) in the arcuate-median eminence region of normal females. Gonadectomized male rats receiving the same ovarian hormone treatment failed to exhibit LH or PRL surges and displayed none of the changes in neurotransmitter turnover or peptide concentrations characteristically seen in the normal female. Unexpectedly however, when females that were treated with estradiol benzoate on days 1–3 postpartum were ovariectomized and treated with ovarian hormones in adulthood, they showed the same accumulation/decline in median eminence NPY concentrations and the same activation of NE and EPI turnover in the arcuate-median eminence region as normal females, even though they showed no LH or PRL surges or changes in median eminence LHRH concentrations. These results suggest that estradiol may not mediate all of the defeminizing actions of androgen exerted during the early neonatal period, and particularly those actions that result in a lack of responsiveness in central noradrenergic, adrenergic and NPY systems in adulthood. However, an action of neonatal estradiol may result in uncoupling of the LHRH neurosecretory system from normal excitatory neurochemical influences.     

Liposomal 1,25 (OH)2 vitamin D3 compounds block proliferation and induce differentiation in myelomonocytic leukaemia cells

The vitamin D₃ derived hormone 1,25 (OH)₂ vitamin D₃ (1,25 D₃) is able to induce growth arrest and differentiation in myelomonocytic leukaemia cells. In order to allow for specific delivery to leukaemic cells the lipophilic compound was incorporated into the lipid membranes of liposomes. Liposomal 1,25 D₃ reduced proliferation as measured by ³H-thymidine incorporation in HL60 leukaemia cells by up to 60%. When liposomes were prepared at different concentrations of 1,25 D₃ 65% inhibition was achieved at 48 n M . The MC 1288 stereoisomer of 1,25 D₃ was more potent and had the same activity at 48 n M .
The effect of the liposomal compounds was specific to myeloid cells as they reduced proliferation in myelomonocytic HL60, monoblastic U937 and monocytic Mono Mac 6 cells but not in the T-cell lines Jurkat and Molt 4.
The antiproliferative effect of liposomal 1,25 D₃ was associated with an induction of differentiation since treated HL60 cells showed a monocytic morphology, increased expression of CD14 and decreased expression of CD33.
When peripheral blood leukaemic cells from M4 and M5 acute myeloid leukaemia (AML) patients were admixed with liposomal compounds an antiproliferative effect was seen in all five cases, including the two cases where free compounds led to enhanced growth. Liposomal delivery of 1,25 (OH)₂ vitamin D₃ may offer a novel approach to treatment of myelomonocytic leukaemia.     

HMBA诱导MEC—1细胞分化角蛋白染色的变化

用六亚甲基二乙酰胺（ＨＭＢＡ）作为分化诱导剂，对人粘液表皮样癌细胞系ＭＥＣ－１细胞中角蛋白进行染色，结果发现高分子量角蛋白在被诱导细胞中含量明显增高。分光光度仪检测角蛋白含量，诱导组ｘ±ｓ为２１８±５１，对照组为１４９±４７，两组有显著差异（Ｐ＜００１），这可能与细胞分化有关。     

Depriving neonatal rats of milk from early lactation has long-term consequences on mammotrope development

The sudden appearance of prolactin-releasing cells during the early postnatal period of the rat is initiated by a small milk-borne peptide. Depriving newborn rats of this early milk factor severely retards mammotrope differentiation during the neonatal period. In the present work, we extend our study of early milk deprivation to the adult. To this end, newborn litters were crossfostered onto mothers that had given birth the same day or one week earlier in order to deprive pups in the latter group of early milk. At 5, 15, and 30 d of age, rats deprived of such milk had decreased percentages of mammotropes (as measured by reverse hemolytic plaque assay, RHPA) when compared to nondeprived animals (P<0.05). By 45 d, the percentage of mammotropes was similar for the two crossfostered groups (P>0.1) and this persisted through d 60. Subsequently, we assessed the secretory capacity of mammotropes from 60-d old rats to secretagogues and found that early milk deprivation had no effect on basal prolactin release (P>0.1), but that it augmented hormone secretion evoked by thyrotropin-releasing hormone (TRH, 100 nM; P<0.01). The inhibitory response to dopamine (DA; 1 μM) and the stimulatory response to angiotensin II (AGII; 100 nM) were not altered by early milk deprivation (P>0.1). Taken together, these results demonstrate that factors in milk from early lactation are required for normal mammotrope differentiation, and that the delay induced by early milk deprivation leads to altered secretory function of mammotropes in adult animals.     

甲状腺机能亢进症辨证分型与甲皱微循环及TT_3、TT_4、FT_4I和吸~(131)碘率的关系

对甲状腺机能亢进症(甲亢)患者进行了临床辨证分型,同步观察甲皱微循环及检测TT_3 TT_4 FT_4I、吸~(131)碘率,探讨它们之间的关系。结果表明甲亢患者微循环积分明显高于对照组(P<0.01),但不同类型甲亢的血淤情况亦不相同。心肝火旺型的微循环积分低于气滞痰凝型和血瘀型,但TT_3、TT_4、FT_4I明显增高;气滞痰凝型居中;血淤型的微循环积分明显增高,但TT_3、TT_4、FT_4I低于其他两型。吸~(131)碘率三型间无差异(P>0.05)。     

Growth and differentiation of adult rat hepatocytes regulated by the interaction between parenchymal and non-parenchymal liver cells

We have devised a medium which supports the continuous growth of hepatocytes without losing their replicative potential and differentiation capacity for a longer period. The medium HCGM, contains four key substances in addition to foetal bovine serum. They are epidermal growth factor, nicotinamide, ascorbic acid 2-phosphate and dimethylsulphoxide. When a non-parenchymal cell fraction containing small hepatocytes and non-parenchymal cells was cultured in HCGM, small hepatocytes grew clonally and differentiated into cells expressing either mature hepatocyte marker proteins or biliary cell marker proteins. Thus, for the first time, we showed the presence of a small compartment of bipotent and highly replicative clonogenic hepatocytes in the rat adult liver. HCGM also supported the growth of stellate cells (Ito cells) which were in the original preparation, suggesting the important role of stellate cells for the successful cultivation of hepatocytes. Together, these results suggest that a microenvironment is produced as a result of cooperative interactions between hepatocytes and stellate cells: one which stimulates the growth and differentiation of clonogenic hepatocytes.     

Tyrosine hydroxylase and serotonin containing cells in embryonic rat rhombencephalon: a whole-mount immunocytochemical study

Rhombencephala from rat embryos were processed as whole-mounts for immunocytochemical detection of monoaminergic cell populations, using antibodies to tyrosine hydroxylase (TH) and serotonin (5-HT). Specific advantages of the whole-mount technique over the classical serial-section method were that even isolated immunoreactive (IR) cells could be detected easily, and three-dimensional relationships could be ascertained without the need for serial reconstruction. Embryos between embryonic days (E) 12 and 16 (the day following nocturnal mating being considered as E1) were used in this study. Both TH and 5-HT immunoreactivities were already detectable at E12, even in the smallest embryos (crown-rump length: 6 mm), but there was a striking difference in the number and regional distribution of these two types of IR cells. TH was expressed in several cell groups located in the rostral rhombencephalon (the presumed anlage of the A4-7 complex) as well as in the caudal rhombencephalon (the presumed anlagen of groups A1-2 and C1-3), whereas 5-HT was expressed in very few cells located near the rostral border of the rhombencephalon (presumed anlage of the B4-9 complex). Although the three-dimensional distribution of the TH-IR cell groups underwent some modifications during the period studied, its general pattern remained relatively stable after E12. This contrasted with the sequential appearance of the 5-HT-IR cell groups and their spatial transformations during this period. Using the rhombencephalic isthmus as a landmark, we found that conspicuous 5-HT-IR fibre bundles penetrated into the mesencephalon from E13 onwards, but that the 5-HT IR cell bodies were exclusively located caudal to the borderline between the mesencephalon and the rhombencephalon (the rhombencephalic isthmus). We therefore suggest the term "rostral rhombencephalic raphe nuclei" for the rostral 5-HT cell groups instead of "mesencephalic raphe nuclei," which is a misnomer. Close spatial association between TH and 5-HT-IR elements was observed mainly in the caudal rhombencephalon, where 5-HT-IR fibres coursed through an area containing numerous TH-IR cell bodies (the presumed anlagen of groups A1-2 and C1-3).     

Radioautographic evidence for slow astrocyte turnover and modest oligodendrocyte production in the corpus callosum of adult mice infused with 3H-thymidine

To find out whether glial cells proliferate in the corpus callosum of adult mice, two series of experiments were carried out. The first one made use of 9-month-old "aged" male mice. Some of them were given 3H-thymidine as a 2-hour pulse to examine which cells became labeled and, therefore, had the ability to divide. Others were sacrificed after a continuous infusion of 3H-thymidine for 30 days to examine whether the label would then appear in different cells. In other aged animals, the 30-day infusion was followed by 60 or 180 days without 3H-thymidine to determine whether cells retained or lost their label with time. A second series of experiments was carried out in 4-month old "young adult" male mice to seek confirmation of the main conclusions. Following the 3H-thymidine pulse given to aged mice, only immature glial cells were labeled. After a 30-day infusion, 12.1% astrocytes and 1.1% oligodendrocytes were labeled, so that the net daily addition rate of astrocytes averaged 0.4% and of oligodendrocytes, 0.04%. In young adult mice, the rate after a 7-day infusion averaged 0.9% for astrocytes and 0.08% for oligodendrocytes. However, when the 30-day infusion into aged mice was followed by 60 and 180 days without 3H-thymidine, the labeled astrocytes decreased to 5.3% and 0%, respectively, whereas the number of labeled oligodendrocytes did not change significantly. The interpretation of the results is that the immature cells present in the corpus callosum of mice continue dividing throughout life and their progeny give rise to astrocytes and oligodendrocytes. In the case of astrocytes, the production of new cells occurs in parallel with a loss, so that the astrocyte population turns over. In the case of oligodendrocytes, there is a small production of new, apparently stable cells.