In vitro differentiation characteristics of cultured human mononuclear cells-implications for endothelial progenitor cell biology

Endothelial progenitor cells (EPCs) have been implicated in the pathogenesis and treatment of cardiovascular disease. By use of quantitative uptake of DiLDL and lectin staining, EPCs have been characterized reliably. However, the exact nature and function of this cell population still remains poorly defined. In an attempt to further clarify the cell surface characteristics of EPCs, mononuclear cells (MNCs) were isolated from human blood and cell surface expression patterns were defined by FACS analysis before and after differentiation for 1-10 days in cell culture. "Classical" double staining for DiLDL and Ulex europaeus increases to 89.2 /- 0.05 after 10 days in culture. Looking at EPC-specific markers by FACS analysis, 0.18 +/- 0.11% of freshly isolated MNCs express CD34, 0.13 +/- 0.08% CD133, 0.59 +/-0.51% VEGFr2, 0.01 +/- 0.02% CD34/VEGFr2, 0.09 +/- 0.05% CD34/CD133, 0.58 +/- 0.13% CD34/CD31, and 0.02 +/- 0.01% CD34/CD146, respectively. Induction of the endothelial phenotype is evidenced by positive staining for VEGFr2, CD146, and CD31, and occurs in co-expression with stem cell markers in less than 2 +/- 0.52% of cultured cells. Expression of CD34 increases to 0.38 +/- 0.10% after 10 days, whereas the CD133(+) cell population shows an initial peak at 24h (0.29 +/- 0.18%) before decreasing to 0.15 +/- 0.02% at day 10. EPCs co-expressing CD34/CD133 increase to 0.19 +/- 0.09% after 10 days, and EPCs double-positive for CD34/VEGFr2 increase to 1.45 +/- 1.03%. Looking at leukocyte, lymphocyte, and monocyte lineage markers, 56.27 +/- 0.15% of freshly isolated MNCs express CD45, 7.13 +/- 0.02% CD14, and 38.65 +/- 0.01% CD3. Over the 10-day culture period, expression of CD45 decreases to 28.48 +/- 0.18%, CD3 to 23.11 +/- 0.02%, and CD14 to 0.09 +/- 0.02%. Cells co-expressing CD3/CD45 decrease from 38.88 +/- 0.33% to 24.86 +/- 2.49% after 10 days in culture. These findings extend present knowledge by showing that human MNCs differentiate at a very low rate to EPCs, while a majority of the cultured cell population remain committed to the leukocyte or lymphocyte lineage. Careful surface marker analysis might be necessary when using in vitro EPC differentiation systems.     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

In vitro neuronal differentiation of cultured human embryonic germ cells

Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1(+), SSEA-3(-), SSEA-4(+), TRA-1-60(+), TRA-1-81(+), Oct-4(+), and hTERT(+), the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro. Furthermore, these derived cells tended to neurally differentiate in vitro, especially under high-density culture conditions. We successfully isolated neural progenitor cells from differentiating hEG cultures and about 10% cells induced by 2microM all-trans-retinoic acid (RA) or 0.1mM dibutyryl cyclic AMP (dbcAMP)/1mM forskolin to mature neurons expressing microtubule-associated protein 2ab (MAP2ab), synaptophysin, beta-tubulin III, neuron-specific enolase (NSE), tyrosine hydroxylase (TH), but no glial fibrillary acid protein (GFAP) and choline acetyl transferase (ChAT). The data suggested that hEG cells may provide a potential source of cells for use in transplantation therapy for neurological degenerative diseases.     

In vitro cultured primary roots derived from stem segments of cassava (Manihot esculenta) can behave like storage organs

BACKGROUND AND AIMS: Cassava (Manihot esculenta) has three adventitious root types: primary and secondary fibrous roots, and storage roots. Different adventitious root types can also regenerate from in vitro cultured segments. The aim of this study was to investigate aspects of in vitro production of storage roots. METHODS: Morphological and anatomical analyses were performed to identify and differentiate each root type. Twenty-nine clones were assayed to determine the effect of genotype on the capacity to form storage roots in vitro. The effects of cytokinins and auxins on the formation of storage roots in vitro were also examined. KEY RESULTS: Primary roots formed in vitro and in vivo had similar tissue kinds; however, storage roots formed in vitro exhibited physiological specialization for storing starch. The only consistent diagnostic feature between secondary fibrous and storage roots was their functional differentiation. Anatomical analysis of the storage roots formed in vitro showed that radial expansion as a consequence of massive proliferation and enlargement of parenchymatous cells occurred in the middle cortex, but not from cambial activity as in roots formed in vivo. Cortical expansion could be related to dilatation growth favoured by hormone treatments. Starch deposition of storage roots formed in vitro was confined to cortical tissue and occurred earlier than in storage roots formed in vivo. Auxin and cytokinin supplementation were absolutely required for in vitro storage root regeneration; these roots were not able to develop secondary growth, but formed a tissue competent for starch storing. MS medium with 5 % sucrose plus 0.54 microM 1-naphthaleneacetic acid and 0.44 microM 6-benzylaminopurine was one of the most effective in stimulating the storage root formation. Genotypes differed significantly in their capacity to produce storage roots in vitro. Storage root formation was considerably affected by the segment's primary position and strongly influenced by hormone treatments. CONCLUSIONS: The storage root formation system reported here is a first approach to develop a tuberization model, and additional efforts are required to improve it. Although it was not possible to achieve root secondary growth, after this work it will be feasible to advance in some aspects of in vitro cassava tuberization.     

Protein biosynthesis in cultured human hair follicle cells

A new technique has been used for culturing human keratinocytes. The cells grow on the basement membrane-like capsules of bovine lenses. Lens cells were removed from the capsules by rigid trypsinization. In order to exclude any contamination with remaining living cells the isolated capsules were irradiated with X-rays at a dose of 10,000 rad. In this way human epithelial cells can be brought in culture from individual hair follicles. Since feeder cells are not used in this culture technique, the biosynthesis of keratinocyte proteins can be studied in these cultures. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble, and a urea-insoluble fraction. Product analysis has been performed on the first two fractions revealing protein patterns identical to those of intact hair follicles. Product analysis of the urea-soluble fractions of microdissected hair follicles shows that the protein pattern of the cultured keratinocytes resembles the protein pattern of the hair follicle sheath. Studies on the metabolism of benzo(a)pyrene revealed that the enzyme aryl hydrocarbon hydroxylase (AHH) is present in cultured hair follicle cells. A possible use of our culture system for eventual detection of inherited predisposition for smoking-dependent lung cancer is discussed.     

Enzyme induction in cultured human hair follicle cells

The enzyme aryl hydrocarbon hydroxylase is present in murine and human cultures of keratinocytes. While the activity of aryl hydrocarbon hydroxylase in murine cultures was found to be inducible after exposure to benz(a)anthracene, human keratinocytes originating from the hair follicle did not respond to this treatment. Hence, cultured human keratinocytes are not suitable for studying the molecular events that mediate the proces of aryl hydrocarbon hydroxylase induction.     

In vitro colony formation by cryopreserved primary human tumor cells

The effect of cryopreservation on the ability of primary human cancer cells to form colonies in a two-layer agar system was examined. Although considerable variation occurred, concentrations of 5 or 10% dimethylsulfoxide employed with slow freezing rates allowed survival of colonies in the range 20-40% or greater of nonfrozen controls. The methods used in this study do not require elaborate freezing equipment, and can be used for the cryopreservation of a wide variety of types of cancers.     

Hypergravity affects cell cycle progression and caveolin-1 expression of in vitro cultured human primary endothelial cells

In hypogravity conditions unloading of skeletal muscle fibres causes alterations in skeletal muscle structure and functions including growth, gene expression, cell differentiation, cytoskeletal organization, contractility and plasticity. Recent studies have identified sphingosine I -phosphate (SPP) as a lipid mediator capable of eliciting intracellular Ca2+ transients, cell proliferation, differentiation, suppression of apoptosis, as well as cell injury repair. The aim of this research is to evaluate a possible involvement of SPP in skeletal muscle cells differentiation and repair from space-flight damage. Particularly, we investigated the Ca2+ sources and the changes on the cytoskeletal rearrangement induced by SPP in a mouse skeletal (C2C12) myoblastic cell line. Confocal fluorescence imaging revealed that SPP elicited Ca2+ transients which propagated throughout the cytosol and nucleus. This response required extracellular and intracellular Ca2+ mobilization. SPP also induced cell contraction through a Ca2(+)- independent/Rho-dependent pathway. The nuclear Ca2+ transients are suggestive for an action of SPP in the differentiation program and damage repair.     

Hypergravity impairs angiogenic response of in vitro cultured human primary endothelial cells

A variety of evidence suggest that cardiovascular system functions are impaired in altered gravity conditions. In this study we investigated the influence of hypergravity environment (3g) on endothelial cell proliferation, endothelial vasoactive compound production and on in vitro angiogenesis. We found that cultured primary human endothelial cells were very sensitive to mild hypergravity conditions. Even if we did not record changes in cell viability and apoptosis, we found significant differences in cell proliferation, prostacyclin (PGI2) synthesis, nitric oxide (NO) synthesis, and in angiogenic responses. Using western blotting technique we detected an increased expression of cycloxygenase-2 (COX-2) in primary endothelial cells exposed for 48 hours to hypergravity, in comparison to those exposed to normal gravity.     

In vitro osteogenesis from human skin-derived precursor cells

Embryonic tissue and organ development are initiated from three embryonic germ layers: ectoderm (skin and neuron), mesoderm (blood, bone, muscle, cartilage and fat) and endoderm (respiratory and digestive tract). In former times, it was believed that cell types in each germ layer are specific and do not cross from one to another throughout life. A new finding is that one tissue lineage can differentiate across to another tissue lineage, and this is termed transdifferentiation. We were interested in studying the transdifferentiation of skin-derived precursor cells (ectoderm layer) to osteoblastic cells (mesoderm layer). Human skin-derived precursor cells (hSKP) were isolated and induced into an osteoblastic lineage using osteogenic induction medium (alpha-MEM plus 10% fetal bovine serum supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone). The specific characteristics of osteoblastic cells, including the expression of enzyme alkaline phosphatase, the deposition of mineral and the expression of osterix, bone sialoprotein and osteocalcin, were detected only from the inductive group. The results in our study show that SKP from human skin are a practically available source for osteogenesis. The samples are easily obtainable for autologous use with a high expansion capacity.     

In vitro culturing and characteristics of transit amplifying epithelial cells from human prostate tissue

The prostatic epithelium is functionally organized in stem cell units. This unit consists of a slow turn over stem cell within the basal epithelial layer which can replenish itself and provide progeny which differentiate down either a neuroendocrine or exocrine pathway. The maturation along the exocrine pathway initially involves transit amplifying cells within the basal layer proliferating and subsequently the progeny maturing into intermediate cells. These intermediate cells migrate into the luminal layer where they terminally differentiate into non-proliferative secretory luminal cells which express prostate specific differentiation markers, like PSA. A growing body of experimental evidence has identified the proliferating transit amplifying/intermediate cells as the cells of origin for the common prostatic adenocarcinomas. Using a series of growth characteristics, and mRNA and protein markers, we have validated that primary cultures can be established in serum free defined media from surgically resected human prostates which are composed of essentially pure population of transit amplifying cells. At each serial passage, the subsequent cultures undergo enhanced maturation into intermediate cells and by the 7-10th passage these cells eventually lose their proliferative ability. This study validates that these cells are a useful and relevant system for the determination of molecular events involved in prostatic carcinogenesis.     

Auditory hair cell explant co-cultures promote the differentiation of stem cells into bipolar neurons

Auditory neurons, the target neurons of the cochlear implant, degenerate following a sensorineural hearing loss. The goal of this research is to direct the differentiation of embryonic stem cells (SCs) into bipolar auditory neurons that can be used to replace degenerating neurons in the deafened mammalian cochlea. Successful replacement of auditory neurons is likely to result in improved clinical outcomes for cochlear implant recipients. We examined two post-natal auditory co-culture models with and without neurotrophic support, for their potential to direct the differentiation of mouse embryonic SCs into characteristic, bipolar, auditory neurons. The differentiation of SCs into neuron-like cells was facilitated by co-culture with auditory neurons or hair cell explants, isolated from post-natal day five rats. The most successful combination was the co-culture of hair cell explants with whole embryoid bodies, which resulted in significantly greater numbers of neurofilament-positive, neuron-like cells. While further characterization of these differentiated cells will be essential before transplantation studies commence, these data illustrate the effectiveness of post-natal hair cell explant co-culture, at providing valuable molecular cues for directed differentiation of SCs towards an auditory neuron lineage.     

In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl₂, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.     

Effects of introducing cultured human chondrocytes into a human articular cartilage explant model

Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue.     

Lectin from Griffonia simplicifolia identifies an immature-appearing subpopulation of sensory hair cells in the avian utricle

Brain Cell Biology - The sensory hair cells of the inner ear are coated with a variety of glycoproteins and glycolipids which can be identified by the binding of specific lectins. The present study...     

In vitro degradation of human hair by Hendersonula toruloidea.

The in vitro ability of 5 Hendersonula toruloidea and 1 control Microsporum gypseum strain to degrade human hair has been studied by analysis of culture medium and microscopy. H. toruloidea was able to utilize human hair as a source of nutrients but the extent varied according to the isolate and in all cases degradation was less marked than with M. gypseum.     

In vitro generation of human cells with cancer stem cell properties

Cancer stem cells (CSCs) have been implicated in the maintenance and progression of several types of cancer. The origin and cellular properties of human CSCs are poorly characterized. Here we show that CSC-like cells can be generated in vitro by oncogenic reprogramming of human somatic cells during neoplastic transformation. We find that in vitro transformation confers stem-cell properties to primary differentiated fibroblasts, including the ability to self-renew and to differentiate along multiple lineages. Tumours induced by transformed fibroblasts are hierarchically organized, and the cells that act as CSCs to initiate and maintain tumour growth are marked by the stage-specific embryonic antigen SSEA-1. Heterogeneous lineages of cancer cells in the bulk of the tumour arise through differentiation of SSEA-1(+) fibroblasts, and differentiation is associated with loss of tumorigenic potential. These findings establish an experimental system to characterize cellular and molecular properties of human CSCs and demonstrate that somatic cells have the potential to de-differentiate and acquire properties of CSCs.