Peptides of normal and variant cells of tobacco

Polypeptides solubilized from established normal and variant cell lines of Nicotiana tabacum L cv “Wisconsin 38” have been analyzed by one-dimensional and two-dimensional gel electrophoresis. There was little variability observed in the polypeptide profile in an established cell line; polypeptides present in different clonal lines of cells, all derived from an initial established cell culture, were very similar, if not identical. However, a large fraction of the observed polypeptides present in cytokinin-habituated cell lines (up to 3.8% of the total polypeptides analyzed by two-dimensional gel electrophoresis) were different from those found in the cytokinin-requiring cells from which they were selected. The habituated nature of the selected cell lines was demonstrated to be epigenetic; tissue cultures that were reisolated from plantlets regenerated from habituated cell lines did require cytokinin. Further observations demonstrate: (1) that epigenetic events that alter a cellular phenotype change the expression of a relatively large number of polypeptides, (2) that a single epigenetic phenotype may be the result of any one of a number of possible patterns of gene expression, and (3) that epigenetic events are not random events.     

Metabolic profiling of Chinese hamster ovary cell cultures at different working volumes and agitation speeds using spin tube reactors

Culture systems based on spin tube reactors have been consolidated in the development of manufacturing processes based on Chinese hamster ovary (CHO) cells. Despite their widespread use, there is little information about the consequences of varying operational setting parameters on the culture performance of recombinant CHO cell lines. Here, we investigated the effect of varying working volumes and agitation speeds on cell growth, protein production, and cell metabolism of two clonally derived CHO cell lines (expressing an IgG1 and a “difficult-to-express” fusion protein). Interestingly, low culture volumes increased recombinant protein production and decreased cell growth, while high culture volumes had the opposite effect. Altering agitation speeds exacerbated or moderated the differences observed due to culture volume changes. Combining low agitation rates with high culture volumes suppressed growth and recombinant protein production in CHO cells. Meanwhile, high agitation rates narrowed the differences in culture performance between low and high working volumes. These differences were also reflected in cell metabolism, where low culture volumes enhanced oxidative metabolism (linked to a productive phenotype) and high culture volume generated a metabolic profile that was predominately glycolytic (linked to a proliferative phenotype). Our findings indicate that the culture volume influence on metabolism modulates the balance between cell growth and protein production, a key feature that may be useful to adjust CHO cells toward a more productive phenotype.     

Induced pluripotent stem (iPS) cells from human fetal stem cells (hFSCs)

Introduction

(1) Human embryonic stem (ES) cells are pluripotent but are difficult to be used for therapy because of immunological, oncological and ethical barriers. (2) Pluripotent cells exist in vivo, i.e., germ cells and epiblast cells but cannot be isolated without sacrificing the developing embryo. (3) Reprogramming to pluripotency is possible from adult cells using ectopic expression of OKSM and other integrative and non-integrative techniques. (4) Hurdles to overcome include i.e stability of the phenotype in relation to epigenetic memory.

Sources of data

We reviewed the literature related to reprogramming, pluripotency and fetal stem cells.

Areas of agreement

(1) Fetal stem cells present some advantageous characteristics compared with their neonatal and postnatal counterparts, with regards to cell size, growth kinetics, and differentiation potential, as well as in vivo tissue repair capacity. (2) Amniotic fluid stem cells are more easily reprogrammed to pluripotency than adult fibroblast. (3) The parental population is heterogeneous and present an intermediate phenotype between ES and adult somatic stem cells, expressing markers of both.

Areas of controversy

(1) It is unclear whether induced pluripotent stem (iPS) derived from amniotic fluid stem cells are fully or partially reprogrammed. (2) Optimal protocols to ensure highest efficiency and phenotype stability remains to be determined. (3) The “level” of reprogramming, fully vs partial, of iPS derived from amniotic fluid stem cells remain to be determined.

Growing points

Banking of fully reprogrammed cells may be important both for (1) autologous and allogenic applications in medicine, and (2) disease modeling.     

Contact inhibition, polyribosomes, and cell surface membranes in cultured mammalian cells

An “overlay” method for rapidly and synchronously inducing contact inhibition in normal cultured cells has been developed. Using this method, disaggregation of cytoplasmic polyribosomes has been observed to occur within a matter of hours after overlay, followed by a decrease in cellular ribosomal RNA. Polysome disaggregation was influenced by the extent of cell-cell interaction and was inhibited by pretreatment of overlay cells with cycloheximide. Treatment of underlay cells with cytosine arabinoside also induced polysome disaggregation, but only after an appreciable lag as compared to that observed in overlaid cultures. Disaggregation could be induced by this method in cultured cells derived from normal tissue but not in cells derived from cancerous tissue. Polysome synthesis in growing “normal” cells (as measured by incorporation of tracer uridine into RNA) was markedly decreased when a cell surface membrane preparation was added to cultures.     

Cellular expansion of MSCs: Shifting the regenerative potential

Mesenchymal-derived stromal or progenitor cells, commonly called “MSCs,” have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.     

A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.     

Characteristics of established KSG cells derived from the scorpionfish Sebastiscus marmoratus: what happens under the hydrostatic pressure like the deep sea?

Advances in cell biology depend, partly, on the development of new cell lines and culture methods. Our research focused on a fibroblast-like cell line, “KSG,” which is derived from scorpionfish fin tissue (Sebastiscus marmoratus). Cells were grown in Leibovitz’s L-15 medium with 10% fetal bovine serum following standard procedures. The optimum growth temperatures for these lines ranged from 15°C to 25°C. All cells survived storage for at least 3 yr at ?80°C. Subsequently, they were continuously cultured until the 78th generation without evident changes in their morphology. Moreover, we were able to culture KSG cells in the absence of fetal bovine serum in a culture medium containing the fish serum “SeaGrow.” Optimum SeaGrow concentrations for these cells ranged from 5% to 20%. The growth rate of KSG cells decreased when the concentration of SeaGrow was reduced to 1%. However, this decrease could be partially reversed by adding 0.5% “Hy-Fish.” In addition, the inclusion of Hy-Fish improved cell adhesion. KSG cells that were cultured in serum-free culture media containing 0.5% and 1% Hy-Fish had been added and were able to survive at low densities. Furthermore, we successfully transfected this cell line with a commercial plasmid vector coding a fluorescent protein using the cationic lipid. Finally, the analyses of cell behavior under hydrostatic pressure showed that some pressures (10 MPa) helped the cells to proliferate more.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. VIII. Expression and shedding of Fcγ receptors on metastatic tumor cell variants

The expression of receptors for the Fc portion of IgG immunoglobin molecules was studied on tumor cell lines with high and low metastatic capacity. Two tumor cell lines from DBA/2 mice that had high metastatic activity, ESb and MDAY-D2, contained a high percentage of Fc receptor positive cells, as detected in a rosette assay with IgG antibody-coated erythrocytes (EA). In contrast, the low metastatic parental line Eb, from which ESb was derived, contained only a low percentage of EA-rosette-forming cells. ESb ascites tumor cells adapted to tissue culture in the presence of 2-mercaptoethanol (2ME) had a high expression of Fc receptors, whereas a cell line adapted to tissue culture in the absence of 2ME had a low expression of Fc receptors. “Soluble” Fc receptors were detectable by their ability to bind to EA and to cause blocking of rosette formation. They were found to be present in fluids from tumor-bearing animals, such as serum and cell-free ascites. Even animals with an ascites tumor of the low-metastatic line Eb contained “soluble” Fc receptors. The results are discussed with regard to their possible significance for tumor metastasis.     

Transforming growth factors (TGFs): Properties and possible mechanisms of action

Transforming growth factors (TGFs) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released hy murine sarcoma virus-transformed cells. The TGFs interact with epidermal growth factor (EGF) cell membrane receptors. TGFs are not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. SGF acts as a tumor promoter in cell culture systems and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGFs by transformed cells and the responses of normal cells to the addition of TGFs to the culture medium raise the possibility that cells “autostimulate” their own growth by releasing factors that rebind at the cell surface. The term “autocrine secretion” has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.     

Xenopatients 2.0: Reprogramming the epigenetic landscapes of patient-derived cancer genomes

In the science-fiction thriller film Minority Report, a specialized police department called “PreCrime” apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called “PreCogs”. We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized “stemotoxic” cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge “chromosome therapies” aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a “reprogramming cure” for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research.     

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro

Background aimsMultipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.MethodsMSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to “rescue” the proliferative capacity of MSCs.ResultshPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS.ConclusionshPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.     

MECHANICAL BEHAVIOR OF PLANT TISSUES AS INFERRED FROM THE THEORY OF PRESSURIZED CELLULAR SOLIDS

The mechanical behavior of plant tissues and its dependency on tissue geometry and turgor pressure are analytically dealt with in terms of the theory of cellular solids. A cellular solid is any material whose matter is distributed in the form of beamlike struts or complete “cell” walls. Therefore, its relative density is less than one and typically less than 0.3. Relative density is the ratio of the density of the cellular solid to the density of its constitutive (“cell wall”) material. Relative density depends upon cell shape and the density of cell wall material. It largely influences the mechanical behavior of cellular solids. Additional important parameters to mechanical behavior are the elastic modulus of “cell walls” and the magnitude of internal “cell” pressure. Analyses indicate that two “stiffening” agents operate in natural cellular solids (plant tissues): 1) cell wall infrastructure and 2) the hydrostatic influence of the protoplasm within each cellular compartment. The elastic modulus measured from a living tissue sample is the consequence of both agents. Therefore, the mechanical properties of living tissues are dependent upon the magnitude of turgor pressure. High turgor pressure places cell walls into axial tension, reduces the magnitude of cell wall deformations under an applied stress, and hence increases the apparent elastic modulus of the tissue. In the absence of turgid protoplasts or in the case of dead tissues, the cell wall infrastructure will respond as a linear elastic, nonlinear elastic, or “densifying” material (under compression) dependent upon the magnitude of externally applied stress. Accordingly, it is proposed that no single tangent (elastic) modulus from a stress-strain curve of a plant tissue is sufficient to characterize the material properties of a sample. It is also suggested that when a modulus is calculated that it be referred to as the tissue composite modulus to distinguish it from the elastic modulus of a noncellular solid material.     

Issues in stem cell plasticity

Experimental biology and medicine work with stem cells more than twenty years. The method discovered for in vitro culture of human embryonal stem cells acquired at abortions or from?surplus” embryos left from in vitro fertilization, evoked immediately ideas on the posibility to aim development and differentiation of these cells at regeneration of damaged tissues. Recently, several surprising observations proved that even tissue‐specific (multipotent) stem cells are capable, under suitable conditions of producing a while spectrum of cell types, regardless, whether these tissues are derived from the same germ layer or not. This ability is frequently called stem cell plasticity but other authors also use different names ‐?non‐orthodox differentiation” or?transdifferentiation”. In this paper we wish to raise several important questions and problems related to this theme. Let us remind some of them: Is it possible to force cells of one‐type tissue to lool and act as cells of another tissue? Are these changes netural? Could these trans‐formations be used to treat diseases? What about the bioethic issue? However, the most serious task “still remains to be soloved ‐ how to detect, harvestand culture stem cells for therapy of certain diseases”.     

Phenotypic dissections of the Blastocladiella emersonii zoospore's developmental choice

A developmentally homogeneous neural crest cell population has been used to assay the role of environmental factors in regulating crest cell differentiation. If cultured on tissue culture plastic, virtually all of the cells of this population differentiate into melanocytes. In contrast, when these cells are cultured for 3 or more days on substrata “conditioned” by somite fibroblasts, the proportion of cells undergoing melanogenesis decreased and the proportion expressing formaldehyde-induced fluorescence (FIF), characteristic of catecholamine-containing cells, increased. For a limited period of culture on somite-conditioned substrata, some cells in the population exhibit both pigment granules and fluorescence. Collagen-coated substrata decreased the number of cells that formed pigment but did not stimulate FIF. In contrast, optimum doses of exogenous cellular fibronectin mimicked the effect of somite-conditioned substrata, suppressing melanogenesis and promoting FIF. Glycosaminoglycan-derivatized substrata (i.e., hyaluronic acid, various chondroitin sulfate preparations, and heparin) did not alter the differentiative homogeneity of the cultured crest cell populations. The choice and expression of phenotype by some members of a cultured crest cell population can, therefore, be affected by environmental stimuli provided in the form of certain substrate-attached macromolecules. We suggest that optimal concentrations of some extracellular matrix components produced by embryonic tissue and normally encountered by migrating crest cells may elicit the expression of FIF in crest cells that would otherwise follow a different developmental pathway.