in Vitro Effect of Methamphetamine on Proliferation, Differentiation and Apoptosis of Adipose Tissue Stem Cells

Purpose: Among abused substances, methamphetamine is a psychostimulant drug widely used recreationally with public health importance. This study investigated the effect of methamphetamine on proliferation, differentiation, and apoptosis of human adipose tissue stem cells (AdSCs). Methods: AdSCs were isolated from human abdominal adipose tissue and were characterized for mesenchymal properties and growth kinetics. MTT assay was undertaken to assess methamphetamine toxicity on proliferation and differentiation properties and apoptosis of hAdSCs. Results: Isolated cells were shown to have mesenchymal properties and a population doubling time (PDT) of 40.1 h. Following methamphetamine treatment, expressions of KI-67 and TPX2 as proliferation genes and Col1A1 and PPARg as differentiation genes decreased. Methamphetamine administration increased the expression of Bax and decreased Bcl-2 genes responsible for apoptosis. Conclusions: Our data suggested when AdSCs were exposed to methamphetamine, it decreased proliferation and differentiation properties of stem cells together with an increase in apoptosis. These findings can be added to the literature, especially when methamphetamine is used recreationally for weight loss purposes.

Full Spectrum Flow Cytometry for High-Dimensional Immunophenotyping of Murine Innate Lymphoid Cells

This 25-parameter, 22-colour full spectrum flow cytometry panel was designed and optimised for the comprehensive enumeration and functional characterisation of innate lymphoid cell (ILC) subsets in murine tissues (Table 1). The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ ILC3 and mature natural killer (NK) cell populations. Further characterisation of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimisation of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin, brefeldin A and pro-ILC activating cytokines.

Characterization of mesenchymal stem cells isolated from Wharton’s jelly of the human umbilical cord

Background Isolation of post-partum umbilical cord Wharton’s jelly stem cells has gained attention as an alternative source of the bone marrow. Because easy isolation, lack of ethical concerns, and the presence of both embryonic and adult stem cells have made them a valuable source for use in therapeutic applications and regenerative medicine. The study utilized a modified protocol using in-house human pooled cord blood serum for isolation and expansion of the mesenchymal stem cells obtained from the human umbilical cord Wharton’s jelly. Cell proliferation and population doubling time and tri-lineage differentiation were assessed, and the expressions of mesenchymal cell surface markers CD44, CD90, CD105, and CD34 were assessed by flow cytometry and RT-PCR. The genetic stability of the isolated cells was assessed by chromosomal karyotype. Results The isolated cells displayed fibroblastic-like morphology and tri-lineage differentiation into adipocyte, chondrocyte, and osteocyte. The isolated cells maintained the proliferative competence with a doubling time ranged from 38 to 42h and corresponded well with the standard positive and negative molecular markers (CD44+, CD90+, CD 105+, and CD34−). Cell senescence occurred at the later passage of the cells (P15) affecting, about 25% of the population. Metaphases spread of the cells showed normal diploid karyotypes, with typical chromosomal plates indicating genetic stability of the isolated cells. Conclusion The primary cultures exhibited success in isolating the umbilical cord Wharton’s jelly mesenchymal stem cells, which maintained their tri-lineage differentiation potential, phenotypes and karyotype characteristics on further passage and expansion.

Counting fluorescently labeled proteins in tissues in the spinning disk microscope using single-molecule calibrations

Quantification of molecular numbers and concentrations in living cells is critical for testing models of complex biological phenomena. Counting molecules in cells requires estimation of the fluorescence intensity of single molecules, which is generally limited to imaging near cell surfaces, in isolated cells, or where motions are diffusive. To circumvent this difficulty, we have devised a calibration technique for spinning-disk confocal (SDC) microscopy, commonly used for imaging in tissues, that uses single-step bleaching kinetics to estimate the single-fluorophore intensity. To cross-check our calibrations, we compared the brightness of fluorophores in the SDC microscope to those in the total-internal-reflection (TIRF) and epifluorescence microscopes. We applied this calibration method to quantify the number of EB1-eGFP proteins in the comets of growing microtubule ends and to measure the cytoplasmic concentration of EB1-eGFP in sensory neurons in fly larvae. These measurements allowed us to estimate the dissociation constant of EB1-eGFP from the microtubules as wells as the GTP-tubulin cap size. Our results show the unexplored potential of single-molecule imaging using spinning disk confocal microscopy and provide a straight-forward method to count the absolute number of fluorophores in tissues which can be applied to a wide range of biological systems and imaging techniques.

ISOLATION OF LIVE CELLS FROM DIFFERENT MICE TISSUES UP TO NINE DAYS AFTER DEATH

Some limited reports suggest that cells can survive in the cadavers for much longer than it was previously thought. In our study we explored how time after death, tissue type (muscle, brain and adipose tissue), storage temperature of cadavers (4 °C or at room temperature) and form of tissue storage (stored as cadavers or tissue pieces in phosphate buffered saline) affect the success of harvesting live cells from mice after death. Cells were isolated from dead tissues and grown in standard conditions. Some cells were used for RNA extraction and RT² Profiler™ PCR Array for cell lineage identification was performed to establish which lineages the cells obtained from post mortem tissues belong to. Results of our study showed that viable cells can be regularly isolated from muscle and brain tissue 3 days post mortem and with difficulty up to 6 days post mortem. Viable cells from brain tissue can be isolated up to 9 days post mortem. No cells were isolated from adipose tissue except immediately after death. In all instances viable cells were isolated only when tissues were stored at 4 °C. Tissue storage did not affect cell isolation. Isolated cells were progenitors from different germ layers. Our results show that live cells could be obtained from mouse cadavers several days after death.Key words: mouse; cadaver; stem cells; brain; muscle; adipose tissue IZOLACIJA ŽIVIH CELIC IZ RAZLIČNIH TKIV MIŠI DO DEVET DNI PO SMRTI Izvleček: Nekatere raziskave kažejo, da je preživetje celic v truplih precej daljše, kot je bilo znano do sedaj. V naši raziskavi smo proučevali, kako na uspešnost izolacije živih celic po smrti miši vplivajo različen čas izolacije po smrti, vrsta tkiva (mišično, možgansko in maščobno), temperatura shranjevanja trupel ter oblika shranjenega tkiva (kot koščki tkiv ali kot celi kadavri). Izolacija in gojenje celic iz tkiv mrtvih miši sta potekali pod standardnimi pogoji. Da bi ugotovili, katerim celičnim linijam pripadajo izolirane celice, je bil del celic uporabljen za izolacijo RNK in nadaljno uporabo v sistemu identifikacije izvornih celičnih linij z verižno reakcijo s polimerazo v realnem času. Rezultati naše raziskave so pokazali, da je žive celice mogoče izolirati iz mišičnega in možganskega tkiva 3 dni po smrti, pogojno tudi do 6 dni po smrti. Iz možganskega tkiva je bilo žive celice mogoče izolirati tudi do 9 dni po smrti. Iz maščobnega tkiva je bilo celice mogoče izolirati zgolj takoj po smrti, ne pa tudi v kasnejših časovnih intervalih. V vseh primerih so bile celice izolirane samo v primeru shranjevanja tkiv pri 4°C. Oblika shranjenega tkiva na izolacijo celic ni vplivala. Izolirane celice so pripadale različnim zarodnim plastem. Rezultati raziskave so pokazali, da je žive celice iz mišjih trupel mogoče izolirati tudi več dni po smrti.Ključne besede: miš; truplo; matične celice; možgansko tkivo; mišično tkivo; maščobno tkivo

A Validation Study on Immunophenotypic Differences in T-lymphocyte Chromosomal Radiosensitivity between Newborns and Adults in South Africa

Children have an increased risk of developing radiation-induced secondary malignancies compared to adults, due to their high radiosensitivity and longer life expectancy. In contrast to the epidemiological evidence, there is only a handful of radiobiology studies which investigate the difference in radiosensitivity between children and adults at a cellular level. In this study, the previous results on the potential age dependency in chromosomal radiosensitivity were validated again by means of the cytokinesis-block micronucleus (CBMN) assay in T-lymphocytes isolated from the umbilical cord and adult peripheral blood of a South African population. The isolated cells were irradiated with 60Co γ-rays at doses ranging from 0.5 Gy to 4 Gy. Increased radiosensitivities of 34%, 42%, 29%, 26% and 16% were observed for newborns compared to adults at 0.5, 1, 2, 3 and 4 Gy, respectively. An immunophenotypic evaluation with flow cytometry revealed a significant change in the fraction of naïve (CD45RA+) T-lymphocytes in CD4+ and CD8+ T-lymphocytes with age. Newborns co-expressed an average of 91.05% CD45RA+ (range: 80.80–98.40%) of their CD4+ cells, while this fraction decreased to an average of 39.08% (range: 12.70–58.90%) for adults. Similar observations were made for CD8+ cells. This agrees with previous published results that the observed differences in chromosomal radiosensitivity between newborn and adult T-lymphocytes could potentially be linked to their immunophenotypic profiles.

KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.

A Novel and Effective Method for Human Primary Skin Melanocytes and Metastatic Melanoma Cell Isolation

The development of an effective method of melanocyte isolation and culture is necessary for basic and clinical studies concerning skin diseases, including skin pigmentation disorders and melanoma. In this paper, we describe a novel, non-enzymatic and effective method of skin melanocyte and metastatic melanoma cell isolation and culture (along with the spontaneous spheroid creation) from skin or lymph node explants. The method is based on the selective harvesting of melanocytes and melanoma cells emigrating from the cultured explants. Thereby, isolated cells retain their natural phenotypical features, such as expression of tyrosinase and Melan-A as well as melanin production and are not contaminated by keratinocytes and fibroblasts. Such melanocyte and melanoma cell cultures may be very useful for medical and cosmetology studies, including studies of antitumor therapies.

A chemically defined biomimetic surface for enhanced isolation efficiency of high-quality human mesenchymal stromal cells under xeno-/serum-free conditions

Mesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by: 1) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell amplification, 2) donor-specific characteristics including MSC frequency/quality that decline with disease state and increasing age, 3) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which pose safety and consistency issues. To overcome those limitations while enabling robust MSC production with constant high yield and quality, we developed a chemically defined biomimetic surface coating, called isoMATRIX, that facilitates the isolation of significantly higher numbers of MSCs in xeno-/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. In sum, the isoMATRIX promotes enhanced xeno-, serum-free, or chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.

Biomedical Applications of Non-Small Cell Lung Cancer Spheroids

Lung malignancies accounted for 11% of cancers worldwide in 2020 and remained the leading cause of cancer deaths. About 80% of lung cancers belong to non-small cell lung cancer (NSCLC), which is characterized by extremely high clonal and morphological heterogeneity of tumors and development of multidrug resistance. The improvement of current therapeutic strategies includes several directions. First, increasing knowledge in cancer biology results in better understanding of the mechanisms underlying malignant transformation, alterations in signal transduction, and crosstalk between cancer cells and the tumor microenvironment, including immune cells. In turn, it leads to the discovery of important molecular targets in cancer development, which might be affected pharmaceutically. The second direction focuses on the screening of novel drug candidates, synthetic or from natural sources. Finally, “personalization” of a therapeutic strategy enables maximal damage to the tumor of a patient. The personalization of treatment can be based on the drug screening performed using patient-derived tumor xenografts or in vitro patient-derived cell models. 3D multicellular cancer spheroids, generated from cancer cell lines or tumor-isolated cells, seem to be a helpful tool for the improvement of current NSCLC therapies. Spheroids are used as a tumor-mimicking in vitro model for screening of novel drugs, analysis of intercellular interactions, and oncogenic cell signaling. Moreover, several studies with tumor-derived spheroids suggest this model for the choice of “personalized” therapy. Here we aim to give an overview of the different applications of NSCLC spheroids and discuss the potential contribution of the spheroid model to the development of anticancer strategies.