Engineering cell alignment in vitro

Cell alignment plays a critical role in various cell behaviors including cytoskeleton reorganization, membrane protein relocation, nucleus gene expression, and ECM remodeling. Cell alignment is also known to exert significant effects on tissue regeneration (e.g., neuron) and modulate mechanical properties of tissues including skeleton, cardiac muscle and tendon. Therefore, it is essential to engineer cell alignment in vitro for biomechanics, cell biology, tissue engineering and regenerative medicine applications. With advances in nano- and micro-scale technologies, a variety of approaches have been developed to engineer cell alignment in vitro, including mechanical loading, topographical patterning, and surface chemical treatment. In this review, we first present alignments of various cell types and their functionality in different tissues in vivo including muscle and nerve tissues. Then, we provide an overview of recent approaches for engineering cell alignment in vitro. Finally, concluding remarks and perspectives are addressed for future improvement of engineering cell alignment.     

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.     

Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses

Background

Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca^2 +/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca^2 +-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca^2 +-CaM complex formation, a CaMKII activator.

Methods

The ability of Trp-His and other peptides to inhibit Ca^2 +-CaM complex formation was investigated by a Ca^2 +-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation.

Results

We showed that Trp-His inhibited Ca^2 +-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca^2 +-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca^2 +-binding site of CaM by forming hydrogen bonds with key Ca^2 +-binding residues of CaM, with a binding free energy of − 49.1 and − 68.0 kJ/mol, respectively.

Conclusions

This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca^2 +-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study.

General significance

The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca^2 +-CaM complex formation in the future.     

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.     

Prevention of in vitro sickling of human erythrocytes by monofunctional amidination

Treatment of sickle erythrocytes with the monoimidate, ethylacetimidate, prevented anoxia-induced sickling in vitro. Hemoglobin isolated from amidated erythrocytes exhibited an increase in the minimum gelling concentration compared to untreated preparations. Although the prevention of sickling did appear to involve chemical modification of hemoglobin S, an increase in the oxygen affinity was not a requisite for the antisickling activity of the imidates. Our results suggest that substitution of amidino for amino groups prevents the aggregation of deoxyhemoglobin S. Furthermore, comparison of monoimidates with bifunctional reagents indicates that molecular crosslinking is not essential for antisickling activity.     

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor β and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.     

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.     

Ultrastructural observations on differentiating human preadipocytes cultured in vitro

A cell type, preadipocytes, isolated from the stroma of adult human adipose tissue appears capable of differentiating, in culture, into a cell with morphological features similar to that observed in terminally differentiated human adipocytes cultured under similar conditions. During this process of differentiation, preadipocytes develop extensive rough endoplasmic reticulum with prominent cisternae, the chromatin of most nuclei becomes decondensed and lipid bodies accumulate to levels observed in cultured adipocytes. Fibroblasts derived from non-adipose tissue do not undergo the same morphological changes when cultured under similar conditions.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Inhibition of the polyadenylation reaction in vitro by polyamines

The effect of polyamines on the polyadenylation reaction in vitro was investigated. Varying concentrations of spermine were added to the reaction catalyzed by purified poly(A) polymerase using rat liver nuclear RNA, poly(A), Escherichia coli tRNA or (Ap)₃A as exogenous primers. The enzyme activity decreased progressively with increasing concentrations of polyamines; complete inhibition was obtained at 0.4 and 1.2 mm spermine for the nuclear RNA- and poly(A)-primed reactions, respectively. No inhibition was observed for the (Ap)₃A-primed reaction. Spermidine and putrescine also inhibited polyadenylation but to a lesser extent than spermine. The degree of inhibition by spermine was related to the polynucleotide primer concentrations. Spermine prevented polyadenylation by binding to the primer but not to the poly(A) polymerase molecule as shown by the migration of [¹⁴C]spermine through glycerol gradients after preincubation with enzyme or tRNA. At concentrations inhibitory to polyadenylation in vitro, spermine could stimulate the DNA-dependent RNA synthesis catalyzed by RNA polymerase II. The present study suggests that low levels of polyamines could be used as specific inhibitors of the poly(A) synthesis in vitro.     

Bilirubin uptake in vitro by the rat intestinal mucosa

Flat sheets of rat jejunum incubated in the presence of unconjugated bilirubin solutions were shown to incorporate bilirubin into the tissue.Bilirubin mucosal uptake, expressed as a function of the incubation time showed a tendency to reach a constant level within 120 min.Solutions of bilirubin in sodium taurocholate gave an incorporation of significantly greater amounts of bilirubin than those prepared with albumin.A structurally similar substance (biliverdin) inhibited bilirubin uptake in a way that suggested competitive inhibition. The results support the view that the mechanism of bilirubin uptake by the rat intestinal mucosa cannot be entirely explained by simple passive diffusion.     

Skin viscoelasticity studied in vitro by microprobe-based techniques

Time-dependent deformation of porcine skin was studied in vitro using specialized microprobe instruments. The deformation behavior of stratum corneum, dermis, and whole skin is examined in the context of results of creep strain, elastic stiffness, and viscoelastic constants obtained in terms of the hold time, loading/unloading rate, and maximum indentation depth (load). Skin time-dependent deformation is significantly influenced by dermis viscoelasticity up to a critical indentation depth (load) beyond which it is controlled by the outermost hard epidermis, particularly stratum corneum. Skin viscoelastic behavior under constant load (creep) and constant displacement (stress relaxation) is interpreted in the light of phenomenological observations and experimental trends.     

Reversible inhibition of cell multiplication in vitro by inhibitors of arginine esteroproteases

Two classes of active-site specific inhibitors of trypsin-like proteases have been shown to inhibit reversibly the multiplication of eukaryotic cells in vitro. The competitive inhibitors p-aminobenzamidine and benzamidine were found to arrest the growth of normal and transformed mouse fibroblasts and human KB cells. The inhibition of cell multiplication occurred within 24 h and was accompanied by substantial decreases in the rates of DNA and protein synthesis. The rate of RNA synthesis was relatively unaffected by the protease inhibitors. In agreement with the known inhibition constants (K_i) for their action against trypsin, p-aminobenzamidine was a much more effective inhibitor of cell multiplication than benzamidine. In addition, tosyllysine chloromethyl ketone (Tos-LysCH₂Cl), an active-site titrant and irreversible inhibitor of trypsin, was found to cause a reversible inhibition of growth. These results suggest that an essential protease activity is necessary for cell multiplication. However, in the case of mouse L-cells, all of the inhibitors and particulary p-aminobenzamidine caused excessive accumulation of lactate in the extracellular medium. This observation, which suggests the possibility of additional sites of action of these compounds in cells, was found to depend upon the cell type and appears to be unrelated to the inhibition of growth.     

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.     

Activation of prothoracic glands by brains in vitro

The effects of brains from both diapausing and non-diapausing Mamestra brassicae pupae on the prothoracic glands from pupae of the same condition were studied by observations of the morphological changes and bioassay of the prothoracic glands in vitro.It was ascertained that the active brains intensified the hormonal activity of prothoracic glands from younger diapausing pupae more than those from older pupae. Further, these results coincided with the fact that the prothoracic glands from brainless pupae were more difficult to activate by active brains the longer the time after the glands had been extirpated.The brains from both younger and older diapausing M. brassicae pupae were able to activate co-cultured inactive prothoracic glands in vitro. These results suggest that even the brain from diapausing pupae of M. brassicae can synthesize and release the prothoracic gland activating hormone in vitro.     

Piperazine derivatives inhibit PrP/PrP propagation in vitro and in vivo

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc^C into a disease-associated isoform PrP^Sc. PrP^Sc accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP^Sc species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP^Sc formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge).     

Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo

Evaluation of the immunogenicity of human mesenchymal stem cells (MSCs) in an allogeneic setting during therapy has been hampered by lack of suitable models due to technical and ethical limitations. Here, we show that allogeneic human umbilical cord blood derived-MSCs (hUCB-MSCs) maintained low immunogenicity even after immune challenge in vitro. To confirm these properties in vivo, a humanized mouse model was established by injecting isolated hUCB-derived CD34+ cells intravenously into immunocompromised NOD/SCID IL2γnull (NSG) mice. After repeated intravenous injection of human peripheral blood mononuclear cells (hPBMCs) or MRC5 cells into these mice, immunological alterations including T cell proliferation and increased IFN-γ, TNF-α, and human IgG levels, were observed. In contrast, hUCB-MSC injection did not elicit these responses. While lymphocyte infiltration in the lung and small intestine and reduced survival rates were observed after hPBMC or MRC5 transplantation, no adverse events were observed following hUCB-MSC introduction. In conclusion, our data suggest that allogeneic hUCB-MSCs have low immunogenicity in vitro and in vivo, and are therefore “immunologically safe” for use in allogeneic clinical applications.