Characterization of human mast cells in long-term culture

Recent studies in rodents have demonstrated that mast cells derived from lymphoid tissues can be grown in longterm culture, provided that supportive growth factors or stromal fibroblasts are added; such findings have not been reported in man. Furthermore, although a hemopoietic origin for mast cells is supported by transplantation studies in mice, the exact origin of the human mast cell or its relationship to the circulating basophil and other hemopoietic cell lineages is unknown. We have investigated the requirements for in vitro growth of human mast cells derived from the infiltrated bone marrow of a patient with systemic mastocytosis, and have characterized both the mast cells proliferating in these cultures and those obtained from splenic infiltrates. Our data approached two questions: (1) Is there any evidence for the origin of mast cells from a bone-marrow-derived stem cell, and, if so, (2) what lineage relationship is there between mast cells and granulopoietic cells, including basophils? First, we have shown the expression of hemopoietic tissue-specific antigens by mast cells, strongly supporting a bone marrow origin for the mast cell in man (at least for those mast cells analyzed here). Second, the complete lack of granulocyte-monocyte markers contrasts with the phenotype of the basophil and suggests that mast cells diverge considerably from other granulopoietic cells during the acquisition of their differentiated specialized functions.     

Interferon: purification and initial characterization from human diploid cells.

Interferon produced by human diploid fibroblast cells in culture has been purified approximately 5000-fold. The purified interferon, when analyzed by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate, contains only one polypeptide component of 20,000 molecular weight. The interferon activity comigrates with this polypeptide, indicating identity of the activity and the polypeptide. Oxidation of this polypeptide with periodic acid and subsequent staining with Fuchsin base indicates that it contains carbohydrate ans suggests that the human fibroblast interferon is a glycoprotein.     

Transdifferentiation of human islet cells in a long-term culture

It has been established that ductal cells or precursor cells within the ductal tree of the pancreas can differentiate into islet cells. Although islet cells can also form exocrine cells, it is unclear whether they arise from precursor (stem) cells or from mature endocrine cells by transdifferentiation. Using a defined culture medium and technique for islet purification, for the first time we were able to maintain human islets in culture for more than a year. Multilabeling immunohistochemical and immunoelectron microscopic examination of the islets at different days of culture using islet cell markers (antibodies to hormones, neuron-specific enolase, chromogranin A) and ductal cell markers (cytokeratins 7 and 19, carbonic anhydrase II, DU-PAN2, CA 19-9, and MUC1) revealed that endocrine cells gradually transdifferentiate to ductal, acinar, and intermediary cells. Although islet hormone secretion ceased after day 28 in culture, endocrine cells were still detectable at day 60. However, later, all endocrine and exocrine cells were replaced by undifferentiated cells that expressed neuron-specific enolase, chromogranin A, laminin, vimentin, cytokeratin 7 and 19, alpha-1-antitrypsin, transforming growth factor-alpha, and epidermal growth factor receptor. Our data thus show that, under proper conditions, human islets can be maintained in vitro over a long period and that, in the culture condition, islet cells seem to transdifferentiate to exocrine cells and undifferentiated cells, which may be considered pancreatic precursor (stem) cells.     

Establishment of human pancreatic ductal cells in a long-term culture

Cultivation and preservation of human pancreatic ductal cells have remained a challenge. With a defined culture medium and refinement of culturing techniques, we have been able to maintain human pancreatic ductal cells without any genetic manipulation in culture for more than 16 months. Freshly isolated ductal fragments were placed on a rocker in M3:5 medium free of collagen for 14 days to remove fibroblasts and endocrine cells before allowing them to attach. The cells produced an excessive amount of mucin and expressed the duct specific cytokeratins (CK) 7 and 19, DU-PAN2, CA19-9, carbonic anhydrase II (CA II), and secretin receptors. During the course of the culture, however, the cells gradually lost the expression of CA II, secretin receptors, DU-PAN2, and CA 19-9 and assumed an undifferentiated phenotype, which showed an upregulation of transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR), an increase in the expression of Ki-67, and an increased binding to Phaseolus vulgaris leucoagglutinin (PHA-L) and tomato lectin. These ductal cells present a useful source with which to study physiologic aspects of ductal cells including differentiation.     

Isolation and characterization of renin-producing human chorionic cells in culture

Chorionic tissue is one of the major extrarenal sites of renin production, and as such, cultured chorionic cells are a potential model for in vitro studies of renin biosynthesis and regulation. Human chorionic cells were isolated from four chorions and maintained in tissue culture for a total of eight subcultures. Total renin production was considerable in the primary cultures, but fell gradually with successive passages. The cells could be frozen and thawed without losing their ability to divide or produce renin. Both the primary cultures and the subcultures contained a single type of elongated cell containing abundant rough endoplasmic reticulum and myofibrils, but no renin granules, suggesting that the cells had smooth muscle-like features. Immunocytochemistry indicated that they contained both renin and prorenin. The renin produced by the chorionic cells was not stored within the cells, but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had biochemical and immunological properties similar to those of pure human renin. The cells contained a renin mRNA that had the same size as that for renal renin (1.6 kilobases), confirming the synthesis of renin by these cells. The cells were also examined for the presence of other components of the renin-angiotensin system. Angiotensinogen and angiotensin I were not detected, but angiotensin-converting enzyme was present in extracts of primary and secondary cultured cells. beta hCG and progesterone were also found in the medium of primary culture. However, the production of beta hCG and progesterone fell after the primary culture, and beta hCG and progesterone were indetectable in secondary and tertiary cultures, respectively. These experiments suggest that these two hormones do not influence renin synthesis or vice versa. Thus, these cultures of human chorionic cells synthesized considerable quantities of prorenin and can provide a permanent source of nonrenal prorenin-producing cells.     

Continuous human bone marrow culture: Ia antigen characterization of probable pluripotential stem cells

The presence of Ia-like antigens on human CFU-C and BFU-e is confirmed and a cell type that lacked immediate capacity for granulocytic colony formation but generated CFU-c after brief incubation in simple suspension culture is identified. This pre-CFU-c, and its immediate progeny, was extremely sensitive to killing by anti-Ia serum with complement. In contrast, anti-Ia serum plus complement treatment of human bone marrow, while eliminating 93%-97% of all CFU-c and BFU-e, did not prevent the rapid regeneration of these progenitor cells and their production for some weeks under the conditions of continuous marrow culture. These studies suggest that the human equivalent of the pluripotential stem cell can replicate for some weeks in culture and generate committed progenitors, such as CFU-c and BFU-e. Furthermore, it would appear that Ia-like antigen is absent on the pluripotential stem cell, is rapidly gained as commitment to the various progenitor cell types occur, and is subsequently lost as these latter undergo differentiation within the marrow.     

High-efficiency gene transfer to human hematopoietic cells maintained in long-term marrow culture

We used a helper-free recombinant retrovirus carrying the neomycin resistance (neor) gene to investigate methods for improving gene transfer efficiencies to clonogenic hematopoietic progenitor cells of human origin and to assess the possibility of gene transfer to the more primitive cells from which clonogenic cells are derived after several weeks in long-term human marrow cultures. The proportion of neor CFU-GM in methylcellulose assays of infected fresh marrow was increased by six- to eightfold (mean 37.4%) by the addition of extra GM colony-stimulating factor and interleukin-1 beta or medium conditioned by a human marrow "stromal" cell line to medium conditioned by agar-stimulated human leukocytes both during the infection and the colony growth period. Similar increases were also noted in the proportion of neor BFU-E, although the efficiencies overall were somewhat lower (up to 25.7%, mean 16.3%). Initiation of long-term cultures with marrow exposed to virus under the same growth factor-supplemented conditions but without any immediate selection step resulted in sustained production of a high proportion of neor CFU-GM and BFU-E for 6 weeks in both the nonadherent and adherent fractions. Molecular analysis was used to confirm the presence of the neo gene after culture. These results demonstrate that stable, high-efficiency gene transfer can be accomplished to the most primitive class of human hematopoietic cells currently detectable that may also have in vivo reconstituting potential. Further use of this approach should provide new insights into human hematopoietic stem cell regulation and allow continued development and assessment of gene therapy procedures.     

Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture

Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, 0°C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as $\text{47.5}\text{35.3}$ kDa, their valences (net protons/molecule) as $\text{2.14}\text{1.85}$. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.     

Continuous long-term culture of human bone marrow

Summary Human bone marrow was cultured with alpha medium, 10^?6m hydrocortisone and a mixture of horse and fetal calf serum in a long-term liquid culture system. An adherent layer was formed which contained fat cells, macrophages, fibroblast-like and epithelioid cells. The layer harboured myeloid cells and their presumptive precursors whilst the nonadherent cells were composed of immature myeloid elements plus mature macrophages and granulocytes whose numbers were maintained for periods of up to 12 weeks. Experiments showed that the use of serum mixtures was superior to horse or fetal calf serum alone and successful culture was accompanied by early growth of the hydrocortisone-dependent fat cells in the adherent layer.     

Isolation, long-term culture, and characterization of rat pancreatic fibroblastoid/stellate cells.

Investigation of pancreatic interstitial fibroblasts has proven difficult in situ. We have established a method for the isolation of pancreatic fibroblastoid/stellate cells by outgrowth from pancreatic tissue explanted into culture dishes. This technique gives a high yield of viable cells from small tissue samples. Outgrown fibroblastoid cells were established as a primary cell line and characterized during long-term culture. We investigated the development of stellate cell markers, i.e. fat storage, expression of desmin, and alpha-smooth muscle actin (alphaSMA), over weeks in culture. AlphaSMA, investigated by indirect immunofluorescence staining and Western blot analysis, revealed a constant rise in expression during routine culture. After 13 passages. approximately 100% of cells were positive for alphaSMA expression, indicating a myofibroblast type of differentiation in vitro.     

Regulation of angiogenic activity of human endometrial endothelial cells in culture by ovarian steroids

Blood vessel growth and regression in human endometrium are regulated throughout the menstrual cycle. We sought a direct role of ovarian steroids on human endometrial endothelial cell (HEEC) proliferation and vascularization. To investigate the HEEC angiogenicity of sex steroids, we developed a reliable method for the isolation of HEEC, which allowed us to investigate the angiogenic effects of sex steroids using immunohistochemistry, immunocytochemistry, Western blot, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt proliferation, and vascular tube formation analyses. We were able to obtain 95-99% pure HEEC with our isolation technique. HEEC expressed predominantly estrogen receptor beta, minimally expressed estrogen receptor alpha, and but did not express progesterone (P(4)) receptors A and B in vivo and in vitro. Estradiol (E(2); 10(-10)-10(-8) m) and P(4) (10(-12)-10(-8) m), alone or in combination, induced HEEC proliferation compared with control values after 48 h of treatment (P < 0.05). Furthermore, after 8 d of treatment, there were significantly more angiogenic patterns in E(2) (10(-8) m), P(4) (10(-10) m), and E(2) plus P(4) (10(-8) and 10(-10) m) treatment groups compared with the control group (angiogenic scores, 2.95 +/- 0.16, 3.26 +/- 0.16, 3.06 +/- 0.17, and 1.93 +/- 0.15, respectively; P < 0.01). In conclusion, our results suggest that there are direct effects of E(2) and P(4) on HEEC and provide a new understanding of the physiological role of sex steroids in the regulation of endometrial events such as angiogenesis.     

Hormonally active arginine-vasopressin suppresses endotoxin-induced fever in rats: lack of effect of oxytocin and a behaviorally active vasopressin fragment

Vasopressin and oxytocin modulate memory processes which effects are dissociated from the typical peripheral endocrine effects of these neuropeptides. Recently, vasopressin has been implicated in the regulation of body temperature. In view of this, experiments were designed to determine whether the antipyretic effect of vasopressin was related to the action of the neuropeptide on memory processes. Fever was induced in rats by intracerebroventricular (i.c.v.) injection of 10 ng bacterial endotoxin (ET), which resulted in a rapid rise in colonic temperature. A second i.c.v. injection 15 min after ET administration of graded amounts (0.01, 0,1, 1 and 100 ng) or arginine-vasopressin (AVP) suppresses ET-induced fever in a dose-dependent manner, 1 ng being the minimally effective amount. Equivalent amounts of des-9-glycinamide-arginine-vasopressin (DG-AVP) or oxytocin (OXT) were ineffective. Large amounts (1,000 ng) of the latter two peptides, however, transiently mimicked the effect of AVP. On one-trial learning passive avoidance behavior, the neurohypophyseal peptides exerted a completely different pattern of effects. AVP and DG-AVP induced a dose-dependent facilitation, while OXT resulted in a dose-dependent attenuation of passive avoidance behavior. These findings suggest that AVP-induced antipyresis is related to the hormonally active AVP and dissociated from the effects of neurohypophyseal hormones and hormone fragments on other CNS processes-like learning and memory.     

A specific growth hormone-binding protein in human plasma: initial characterization

Human (h) GH in plasma exists as a series of size isomers, which are in part explained by the presence of hGH oligomers. However, certain aspects of circulating large mol wt hGH, such as its high relative proportion compared to that in the pituitary, are poorly understood. To explore whether binding of hGH to plasma protein(s) could contribute to the phenomenon of large mol wt hGH, we incubated freshly prepared monomeric [125I]hGH or biosynthesized [3H]hGH with normal human plasma or serum at pH 7.4 for various time periods at 22 and 37 C. Plasma radioactive hGH patterns were then analyzed simultaneously with unincubated tracer hGH by Sephadex G-100 and G-200 chromatography. We found that part of the radioactivity was converted to a component with an apparent mol wt of 85,000, suggesting binding to a plasma protein(s). This phenomenon was inhibited in a dose-dependent fashion by unlabeled hGH. Saturation/Scatchard analysis indicated an association constant (Ka) of 2-3 X 10(8) M-1 and a maximum binding capacity of 20 ng hGH/ml plasma. Binding was rapid, reversible, and specific. A number of polypeptide hormones, including human placental lactogen, hPRL and rat GH, did not inhibit hGH binding. However, the 20K variant of hGH interacted weakly with the plasma binding component (Ka, 1.2 X 10(7) M-1; maximum binding capacity, 450 ng/ml). The binding component was heat labile and could be partially purified by gel permeation chromatography and affinity chromatography on a hGH-Sepharose column. Its estimated mol wt is 60,000-65,000, and it appears to bind one molecule of hGH to form a complex of 80,000-85,000 mol wt. The binding component is neither albumin nor an immunoglobulin. Under physiological conditions, a minimum of 15-18% of circulating hGH is presumably bound to this plasma component. We conclude that a specific, high affinity, low capacity binding protein for hGH with mol wt of 60,000-65,000 exists in normal and hypopituitary human plasma. hGH complexed with this protein forms part of big-big hGH. The biological significance of this binding protein remains to be investigated.     

Long-term culture and functional characterization of follicular cells from adult normal human thyroids.

We have obtained long-term cultures of differentiated proliferating follicular cells from normal adult human thyroid glands. In vitro growth of such human cells has been sustained by a modified F-12 medium, supplemented with bovine hypothalamus and pituitary extracts and no added thyrotropin. Cultures have been expanded, cloned, frozen, successfully retrieved, and characterized. Functional characterization of these cells shows constitutive thyroglobulin production and release and thyrotropin-dependent adenosine 3',5'-cyclic monophosphate production, the latter apparently not associated with significant increases in DNA synthesis or cell proliferation. Genetic characterization of these cells by chromosome counting showed the normal diploid chromosome number. The ability to cultivate differentiated human thyroid follicular cells in long-term culture opens possibilities for investigating the transduction pathways of thyrotropin stimulation in normal and pathological human tissues, developing clinically relevant in vitro assays, and considering cellular and molecular therapies.     

Adenoviral vector-mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34(+) cells and primary colony forming unit-cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34(+) cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P <.01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.     

Microvascular endothelial cells from human omental tissue: modified method for long-term cultivation and new aspects of characterization

A method for long-term cultivation of large amounts of human microvascular endothelial cells from the omental tissue (human omental tissue microvascular endothelial cells, HOTMECs) was devised. The method originally described by Kern, Knedler, and Eckel was modified: HOTMECs were isolated by enzymatic dissociation with collagenase. For primary cultivation and passages, HOTMECs were plated either onto fibronectin-coated petri dishes or onto a human fibroblast extracellular matrix (HFB-ECM) prepared from the same tissue. Omental tissue (10-15 g) yielded 4-8 X 10(5) HOTMECs; more than 90% of the cells adhered to precoated dishes and grew in Waymouth's culture medium supplemented with 20% heat-inactivated fetal calf serum. Confluence was reached 3-5 days after seeding with an average of 1-2 X 10(6) cells/dish. Confluent HOTMEC layers were subcultured at a split ratio of 1:3 up to 11 passages by plating the cells onto dishes coated with HFB-ECM and maintained in long-term culture for up to 3 months. The endothelial origin of these cells was demonstrated as follows. The cells in culture showed the typical "cobblestone" growth pattern and synthesized von Willebrand factor (vWF) as determined by metabolic labeling. Using an indirect immunostaining technique, the cytoplasm of the HOTMECs stained for vWF. A monoclonal antibody specific for human endothelial cells bound exclusively to the cultured cells. The expression of thrombomodulin on the surface of the cultured cells was demonstrated by the activation of protein C by thrombin. In control experiments, these features could be detected on neither fibroblasts nor mesothelial cells.     

An optimal culture condition maintains human hepatocyte phenotype after long-term culture.

BACKGROUND: Long-term culture of primary hepatocytes from various species is impeded by a decrease of cell viability and a loss of hepatocyte-specific function. The aim of the present study was to investigate whether our optimal culture condition (OC) can maintain the phenotype of primary hepatocytes in long-term culture. METHODS: Primary human hepatocytes were cultured in either hepatocyte maintenance medium (HM) or OC for 2-4 weeks. Expression of hepatocyte-specific genes was determined by real-time quantitative RT-PCR. RESULTS: The level of albumin mRNA in human hepatocytes cultured in OC was 11-fold more than in HM and gene expression levels of alpha1-antitrypsin and transferrin were at approximately 40 and 11% of freshly isolated primary human hepatocytes. Electron microscopy revealed that cells in OC displayed hepatocyte properties (e.g. polarity, junctional complexes, bile canaliculi and glycogen particles). Cytochrome P4501A1/2 activity of hepatocytes cultured in OC was 15- and 17-fold higher than in HM at 2 and 4 weeks of culture, and DNA synthesis was higher. CONCLUSIONS: Using our optimal culture condition, we were able to maintain the phenotype of primary human hepatocytes in long-term culture. They not only maintain better liver-specific function, but also retain higher proliferative potential.