Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56⁺ cells grew rapidly, a population of CD15⁺ cells emerged, partly from CD56⁺ cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56⁺ and CD15⁺ cells shared osteogenic and chondrogenic abilities, while CD56⁺ cells presented a myogenic capacity and CD15⁺ cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

Further study of factors affecting amino acid incorporation into protein by isolated mitochondria

1. The incorporation of amino acids into protein in isolated mitochondria has now been studied in more detail, and with mitochondria from a range of tissues. 2. Liver mitochondria from newborn rats are twice as active as those from adult rats. 3. The mitochondria are inactivated by excessive homogenization and repeated freezing and thawing. 4. The incorporation is sensitive to conditions of incubation and in particular to the rate of oxygenation, shape of vessel and depth of fluid. Best results are obtained by incubation in flat-bottomed vessels containing suspensions with less than 3mm. depth of fluid. 5. The requirement for oxidizable substrates has been examined with a range of substances, and most of the common energy-yielding metabolites of the mitochondrion are effective. Their activity is greatly influenced by concentration, and some, but not all, of the substrates show optimum concentrations for incorporation with decreased activity at higher concentrations. 6. Some amino acids can act as energy sources for the incorporation. 7. The effect of increasing the concentration of labelled amino acid is different for different amino acids, and complex effects occur on the addition of amino acid mixtures. 8. It is concluded that the amino acid incorporation into protein finally obtained is the result of many interacting factors related to the structural and metabolic state of the mitochondrion.     

Expression profiles of nestin in vascular smooth muscle cells in vivo and in vitro

Nestin is an intermediate filament protein expressed in neural and mesenchymal stem cells. Here, we investigated the expression of nestin in vascular smooth muscle cells (VSMCs) in vivo and in vitro. In the developing arteries, medial VSMCs were found to express nestin; its expression was prominent in embryos but was down-regulated after birth (3-6 weeks) in a region-dependent manner; its expression was abolished in the adult. Thus, the expression of nestin is specific to developing VSMCs. In primary VMSC cultures, nestin expression was induced by serum, but was independent of cell-cycle progression. Signaling analyses revealed that the serum-induced nestin expression depended on the extracellular signal-regulated kinase (ERK) and protein kinase B (PKB)(Akt) pathways, via the platelet derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Nestin expression was closely related to the up-regulation and activation of Sp1 and Sp3. Among major serum growth factors and cytokines, PDGF-BB was the most potent inducer of nestin expression. Nestin was also up-regulated in arteries undergoing vascular remodeling following balloon injury. Its expression was particularly strong in the cells lining the lumen of the neointima, suggesting a possible correlation between nestin expression and the progression of vascular remodeling.     

Differential integrin expression in pre-implantation embryos developing under in vivo and in vitro conditions

Implantation failure is a major problem in human assisted reproduction, which persists regardless the optimization of endometrial receptivity and selection of genetically and morphologically healthy embryos. Since embryo-endometrium interaction depends on cell junctional, cell adhesion and cell-substratum adhesion molecules, the present study inquired whether in vitro growing murine embryos display similar to the in vivo growing embryos patterns of adhesion molecules. To this extend aVb3 expression and distribution in zygotes and 2-cell stage embryos were studied. The results demonstrated that only the in vivo growing embryos displayed specifically polarized aVb3 distribution, indicating their potential successful interaction with endometrium. Based on previous studies showing that L-carnitine (L-Cn) could affect embryonic development, it was demonstrated that the addition of L-Cn to the culture medium, could lead the in vitro growing embryos to acquire aVb3 expression and distribution similar to the in vivo growing embryos. Visualization of the effect of L-Cn using third harmonic generation imaging showed decreased lipid droplet levels in 2-cell-stage embryos, observation that correlates with an active energetic state of the growing embryos. Thus, the application of L-Cn to the culture medium could assist pre-implantation-state embryos to acquire aVb3 expression and distribution similar to the in vivo developing conditions.     

Effects of the acid polysaccharide fraction isolated from a cultivated Cordyceps sinensis on macrophages in vitro

The acid polysaccharide fraction (APSF) extracted from the mycelia of cultivated Cordyceps sinensis is water-soluble polysaccharide. In this study we evaluated the modulating effects of APSF on murine macrophage cell line RAW264.7. Phagocytotic assay by neutral red and FITC-dextran internalization showed that APSF stimulated the phagocytosis of macrophages. The nitrite levels in the culture supernatant determined using Griess reagent revealed the elevation of NO production after treatment with APSF. RT-PCR and immunocytochemistry assay indicated that APSF promoted both the mRNA and protein expressions of inducible nitric oxide synthase (iNOS). Furthermore, Western blotting demonstrated that NF-κB levels in nucleuses increased after APSF treatment, suggesting that APSF probably stimulated macrophage activities by activating the IκB-NF-κB pathway.     

Regulatory light chains modulate in vitro actin motility driven by skeletal heavy meromyosin

Phosphorylation and Ca²⁺-Mg²⁺ exchange on the regulatory light chains (RLCs) of skeletal myosin modulate muscle contraction. However, the relation between the mechanisms for the effects of phosphorylation and metal ion exchange are not clear. We propose that modulation of skeletal muscle contraction by phosphorylation of the myosin regulatory light chains (RLCs) is mediated by altered electrostatic interactions between myosin heads/necks and the negatively charged thick filament backbone. Our study, using the in vitro motility assay, showed actin motility on hydrophilic negatively charged surfaces only over the HMM with phosphorylated RLCs both in the presence and absence of Ca²⁺. In contrast, good actin motility was observed on silanized surfaces (low charge density), independent of RLC phosphorylation status but with markedly lower velocity in the presence of Ca²⁺. The data suggest that Ca²⁺-binding to, and phosphorylation of, the RLCs affect the actomyosin interaction by independent molecular mechanisms. The phosphorylation effects depend on hydrophobicity and charge density of the underlying surface. Such findings might be exploited for control of actomyosin based transportation of cargoes in lab-on-a chip applications, e.g. local and temporary stopping of actin sliding on hydrophilic areas along a nanosized track.     

Acetate transport by locust rectum in vitro

Everted rectal sacs of Schistocerca gregaria absorb ¹⁴C-acetate from the lumen side at high rates against large electrical and often small concentration differences. Most of the ¹⁴C-activity in the absorbed fluid remains as acetate, but small amounts serve as substrate for aerobic respiration within this tissue. When acetate is substituted for SO₄^?2 or Cl^? in external salines, both short-circuit current (I_sc) and the open-circuit transepithelial potential (PD) increase by as much as 2- to 3-fold. The stimulatory effect of acetate on I_sc and PD exhibits saturation kinetics. The ‘steady-state’ influx of ¹⁴C-acetate from lumen (L) to haemocoel (H) side greatly exceeds efflux (haemocoel to lumen) across short-circuited recta. Over the whole range of acetate concentrations tested, the resulting net flux of acetate is sufficient to explain all of the increase in I_sc caused by this organic anion. Acetate was detected in moderate concentrations in body fluids of locusts. The possible significance of acetate transport in vivo is discussed.     

Thymidine accumulation by choroid plexus in vitro

In vitro, the accumulation and release of [methyl-³H]thymidine ([${}^3\text{H}$]thymidine) by the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of [${}^3\text{H}$]thymidine in the medium of 1.0 μm (or greater), the choroid plexus accumulated [${}^3\text{H}$]thymidine against a concentration gradient by a process that depended on intracellular energy production but did not depend on intracellular binding or metabolism of the [${}^3\text{H}$]thymidine. This transport process was inhibited (although differentially) by various nucleosides and low temperatures but not by 2-deoxyribose or pyrimidine bases. With concentrations of less than 1.0 μm [${}^3\text{H}$]thymidine in the medium, the choroid plexus accumulated [${}^3\text{H}$]thymidine against a concentration gradient. However, the majority of the [${}^3\text{H}$]thymidine within the choroid plexus was metabolized to [${}^3\text{H}$]thymidine nucleotides at low extracellular [${}^3\text{H}$]thymidine concentrations (3 nm). This accumulation process depended, in large part, on saturable intracellular phosphorylation. Thymidine was the principal form released from choroid plexuses that had been incubated for various times in media containing concentrations of thymidine from 3 to 1.0 mm. The release of thymidine from choroid plexus was depressed by cold temperatures and a very high (2.56 mmol/kg) intracellular thymidine concentration.     

In vitro amino acid incorporation into myosin by free polysomes of rat skeletal muscle

Rat skeletal muscle polysomes were separated into free and membrane-bound fractions by centrifugation through 2M sucrose. About 80% of total ribosomes extracted were recovered as free polysomes. Sucrose gradient experiments showed similar size distribution patterns for both free and bound polysomes. Chromatographic and electrophoretic analyses of proteins in the cell free amino acid incorporation system indicated that free polysomes are capable of synthesizing myosin.     

Encapsulation reactions in vitro by haemocytes of Heliothis virescens

A simple in vitro system for studying capsule formation by Heliothis virescens haemocytes was devised. The system produced capsules morphologically and ultrastructurally similar to those formed in vivo. Encapsulation proceeded normally when melanization was inhibited and when divalent cations were absent. Capsule development took place in two physiologically distinct phases. Aggregation of haemocytes around a foreign object (phase 1) was a passive process. Consolidation of haemocytes into a smooth, adherent capsule (phase 2) required metabolic energy. Phase 1 was inhibited irreversibly by propranolol and caffeine. Inhibition of phase 1 by mild trypsinization could be reversed by haemolymph lysate. Preliminary evidence indicates that encapsulation promoting factors in the lysate originate in haemocytes.     

Time studies of ecdysone-action on in vitro apolysis of Chilo suppressalis integument

The relationship between induction of in vitro apolysis and the duration of hormone treatment, and the effects of metabolic inhibitors on the ecdysone-induced apolysis were investigated in the cultured integument taken from the rice stem borer larva, Chilo suppressalis. When fragments of integument were subjected to 0.3 μg/ml β-ecdysone for more than 5 hr and then transferred to hormone free medium, they were induced to apolyse one day after treatment. If the fragments of integument were treated with hormone for 1 to 4 hr at first and then treated with hormone for 2 to 5 hr again after a 5 day interval in hormone free medium, almost all the fragments were induced to apolyse one day after treatment. This result suggests that the action of β-ecdysone on the cultured integument is accumulative. If the fragments of integument were cultured in the medium containing actinomycin-D and then transferred to medium containing β-ecdysone, a strong inhibitory effect on the apolysis of the integument was observed. Similarly, an inhibitory effect appeared when fragments of integument were treated first with hormone and then with puromycin. These results show that the m-RNA synthesis necessary for apolysis was completed within 6 hr after hormone treatment. However, the protein synthesis required for apolysis was not. The relationship of the results obtained from these in vitro experiments to the mode of action of ecdysone is discussed.