Magnetic-activated Cell Sorting Research Articles

The inhibitory effect and mechanism of Yi-qi-hua-yu-jie-du decoction on the drug resistance of gastric cancer stem cells based on ABC transporters

BackgroundThe drug resistance of tumor stem cells is an obstacle in gastric cancer (GC) treatment and the high expression of ABC transporters is a classic reason for drug resistance. This study aimed to construct a reliable GC drug-resistant stem cell model and explore the inhibitory effect and mechanism of Yi-qi-hua-yu-jie-du medicated serum (YQHY) on the drug resistance of GC stem cells based on ABC transporters.MethodsThe tumor stemness biomarker CD44 was primary identification from WGCNA. The magnetic-activated cell sorting (MACS) method was used to separate CD44( +)BGC823/5-Fu (BGC823/5–Fu-CSCs) cells and the stemness characteristics were verified from multiple dimensions. Then, the drug resistance index and expression of ABC transporter genes MDR1 and MRP1 were detected in CD44(−)/CD44(+) cells. The inhibition and apoptosis rates of the cells administrated with YQHY or/and 5-Fu were calculated to confirm that YQHY can suppress the drug resistance of BGC823/5-Fu-CSCs. Afterwards, the effects of YQHY on the expression of MDR1 and MRP1 and the activation of the PI3K/Akt/Nrf2 pathway were observed. Finally, under the administration of IGF-1 (the activator of PI3K/Akt pathway) and Nrf2 siRNA, the mechanism of YQHY on reversing the drug resistance of BGC823/5–Fu-CSCs through inhibiting the expression of MDR1 and MRP1 via PI3K/Akt/Nrf2 was verified.ResultsCD44 was a reliable GC stemness biomarker and can be applied to construct the drug-resistant GC stem cell model CD44(+)BGC823/5-Fu. The growth rate, cell proliferation index, soft agar colony formation, expression of stemness specific genes and tumorigenesis ability of CD44(+)BGC823/5-Fu cells were significantly higher than those of CD44(−)BGC823/5-Fu cells. BGC823/5–Fu-CSCs exhibited strong drug resistance to 5-Fu and high expression of ABC transporter genes MDR1 and MRP1 compared to CD44(-) cells. YQHY increased the inhibition and apoptosis rates to efficiently inhibit the drug resistance of BGC823/5–Fu-CSCs. Meanwhile, it suppressed the expression of MDR1 and MRP1 and restrained the activation of PI3K/Akt/Nrf2 signaling pathway. Finally, it was found that IGF-1 partially restored the activation of PI3K/Akt/Nrf2 pathway, alleviated the inhibition of MDR1 and MRP1, blocked the proliferation-inhibitory and apoptosis-promotion effects. YQHY and si-Nrf2 synergistically suppressed the MDR1/MRP1 expression and the drug resistance of BGC823/5–Fu-CSCs.ConclusionsCD44 was a reliable GC stemness biomarker, and the high expression of ABC transporter genes MDR1 and MRP1 was an important feature of drug-resistant stem cells. YQHY inhibited the MDR1 and MRP1 expression via PI3K/Akt/Nrf2 pathway, thus reversing the drug resistance of BGC823/5–Fu-CSCs.

Isolation of Swine Bone Marrow Lin-/CD45-/CD133 + Cells and Cardio-protective Effects of its Exosomes

BackgroundThe identification in murine bone marrow (BM) of CD133 + /Lin-/CD45- cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the swine BM. Heart failure is the terminal stage of many cardiovascular diseases, and its key pathological basis is cardiac fibrosis (CF). Research showed that stem cell derived exosomes may play a critical role in cardiac fibrosis. The effect of exosomes (Exos) on CF has remained unclear.ObjectiveTo establish an isolation and amplification method of CD133 + /Lin-/CD45- cells from newbron swine BM in vitro, explore an highly efficient method to enrich swine bone marrow derived CD133 + /Lin-/CD45- cells and probe into their biological characteristics further. Furher more, to extract exosomes from it and explore its effect on CF.MethodsThe mononuclear cells isolated from swine bone marrow by red blood cell (RBC) lysing buffer were coated by adding FcR blocking solution and coupled with CD133 antibody immunomagnetic beads, obtaining CD133 + cell group via Magnetic Activated Cell Sorting (MACS). In steps, the CD133 + /Lin-/CD45- cells were collected by fluorescence-activated cell sorting (FACS) labeled with CD133, Lin and CD45 antibodies, which were cultured and amplified in vitro. The biological features of CD133 + /Lin-/CD45- cells were studied in different aspects, including morphological trait observed with inverted microscope, ultrastructural characteristics observed under transmission electron microscope, expression of pluripotent markersidentified by immunofluorescent staining and Alkaline phosphatase staining.The Exos were extracted using a sequential centrifugation approach and its effects on CF were analyzed in Angiotensin II (Ang-II) induced-cardiac fibrosis in vivo. Rats in each group were treated for 4 weeks, and 2D echocardiography was adopted to evaluate the heart function. The degree of cardiac fibrosis was assessed by Hematoxylin–Eosin (HE) and Masson's trichrome staining.ResultsThe CD133 + /Lin-/CD45- cells accounted for about 0.2%-0.5% of the total mononuclear cells isolated from swine bone marrow. The combination of MACS and FACS to extract CD133 + /Lin-/CD45- cells could improved efficiency and reduced cell apoptosis. The CD133 + /Lin-/CD45- cells featured typical traits of pluripotent stem cells, the nucleus is large, mainly composed of euchromatin, with less cytoplasm and larger nucleoplasmic ratio, which expressed pluripotent markers (SSEA-1, Oct-4, Nanog and Sox-2) and alkaline phosphatase staining was positive.Animal experiment indicated that the cardiac injury related indexes (BNP、cTnI、CK-MB and TNF-α), the expression of key gene Smad3 and the degree of cardiac fibrosis in Exo treatment group were significantly reduced compared with the control group. 4 weeks after the treatment, cardiac ejection fraction (EF) value in the model group showed a remarkable decrease, indicating the induction of HF model. While Exo elevated the EF values, demonstrating cardio-protective effects.ConclusionThe CD133 + /Lin-/CD45- cells derived from swine bone marrow were successfully isolated and amplified, laying a good foundation for further research on this promising therapeutic cell. The Exos may be a promising potential treatment strategy for CF.Graphical

Genetic and Epigenetic Evaluation of Human Spermatogonial Stem Cells Isolated by MACS in Different Two and Three-Dimensional Culture Systems.

Objective Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of this study is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy in propagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS). Materials and Methods IIn this experimental study, obstructive azoospermia (OA) patient-derived spermatogonial cells were divided into two groups. The MACS enriched and non-enriched spermatogonial stem cells (SSCs) were cultured in the control and treated groups; co-culture of SSCs with Sertoli cells of men with OA, co-culture of SSCs with healthy Sertoli cells of fertile men, the culture of SSCs on PLA nanofiber and culture of testicular cell suspension. Gene-specific methylation by MSP, expression of pluripotency (NANOG, C-MYC and OCT-4), and germ cells specific genes (Integrin α6, Integrin β1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse. Results The use of MACS for the purification of human stem cells was effective at about 69% with the culture of the testicular suspension, being the best culture system. Upon purification, the germ-specific gene expression was significantly higher in testicular cell suspension and treated groups (P≤0.05). During the culture time, gene-specific methylation patterns of the examined genes did not show any changes. Our data from transplantation indicated the homing of the donor-derived cells and the presence of human functional sperm. ConclusionOur in vivo and in vitro results confirmed that culture of testicular cell suspension and selection of spermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells. The epigenetic patterns showed that the specific methylation of the evaluated genes at this stage remained constant with no alteration throughout the entire culture systems over time.

Somatic GJA4 gain-of-function mutation in orbital cavernous venous malformations

Orbital cavernous venous malformation (OCVM) is a sporadic vascular anomaly of uncertain etiology characterized by abnormally dilated vascular channels. Here, we identify a somatic missense mutation, c.121G > T (p.Gly41Cys) in GJA4, which encodes a transmembrane protein that is a component of gap junctions and hemichannels in the vascular system, in OCVM tissues from 25/26 (96.2%) individuals with OCVM. GJA4 expression was detected in OCVM tissue including endothelial cells and the stroma, through immunohistochemistry. Within OCVM tissue, the mutation allele frequency was higher in endothelial cell-enriched fractions obtained using magnetic-activated cell sorting. Whole-cell voltage clamp analysis in Xenopus oocytes revealed that GJA4 c.121G > T (p.Gly41Cys) is a gain-of-function mutation that leads to the formation of a hyperactive hemichannel. Overexpression of the mutant protein in human umbilical vein endothelial cells led to a loss of cellular integrity, which was rescued by carbenoxolone, a non-specific gap junction/hemichannel inhibitor. Our data suggest that GJA4 c.121G > T (p.Gly41Cys) is a potential driver gene mutation for OCVM. We propose that hyperactive hemichannel plays a role in the development of this vascular phenotype.

Double-negative T cells mediate M1 polarization of microglial cells via TNF-α-NLRP3 to aggravate neuroinflammation and cognitive impairment in Alzheimer's disease mice.

We mainly study the role and regulatory mechanism of double-negative T cells(DNTs) in Alzheimer's disease(AD). The mice splenic DNTs were separated and amplified by Rosettesep antibody adsorption method and Easysep magnetic activated cell sorting. DNTs were intraperitoneally injected into the APP/PS1-AD mice model, which was found to aggravate cognitive impairment in mice. DNTs secreted tumor necrosis factor α (TNF-α) to promote the activation of NLRP3 and the M1 polarization of microglial cells, and silencing NLRP3 with small interfering RNA (siRNA) suppressed the effect of DNTs. DNTs were later cocultured with mice microglial cell line BV2, then fluorescence staining was conducted to detect NLRP3 expression, and enzyme-linked immunoassay was performed to measure the expression of inflammatory factors. Moreover, the levels of NLRP3, ASC, and TNFR1 proteins were detected by western-blot assay, and the proportion of F4/80 + CD11b + M1 cells was detected by flow cytometry. DNTs promoted the M1 polarization of BV2 cells and the activation of NLRP3 inflammasome. After treatment of BV2 cells with NLRP3 inhibitor, the effect of DNTs was weakened. Later, TNF-α siRNA was transfected into DNTs, and it was found that DNTs with TNF-α silencing had markedly weakened polarization effect on BV2 cells. We discovered that the proportion of DNTs increased in AD patients. DNTs secreted TNF-α to regulate the activation of NLRP3 inflammasome and the M1 polarization of microglial cells, thus promoting the central inflammatory response and aggravating the cognitive impairment in AD mice.

Angiogenic gene characterization and vessel permeability of dermal microvascular endothelial cells isolated from burn hypertrophic scar

Hypertrophic scar (HTS) formation is a common challenge for patients after burn injury. Dermal microvascular endothelial cells (DMVECs) are an understudied cell type in HTS. An increase in angiogenesis and microvessel density can be observed in HTS. Endothelial dysfunction may play a role in scar development. This study aims to generate a functional and expression profile of HTS DMVECs. We hypothesize that transcript and protein-level responses in HTS DMVECs differ from those in normal skin (NS). HTSs were created in red Duroc pigs. DMVECs were isolated using magnetic-activated cell sorting with ulex europaeus agglutinin 1 (UEA-1) lectin. Separate transwell inserts were used to form monolayers of HTS DMVECs and NS DMVECs. Cell injury was induced and permeability was assessed. Gene expression in HTS DMVECS versus NS DMVECs was measured. Select differentially expressed genes were further investigated. HTS had an increased area density of dermal microvasculature compared to NS. HTS DMVECs were 17.59% less permeable than normal DMVECs (p < 0.05). After injury, NS DMVECs were 28.4% and HTS DMVECs were 18.8% more permeable than uninjured controls (28.4 ± 4.8 vs 18.8 ± 2.8; p = 0.11). PCR array identified 31 differentially expressed genes between HTS and NS DMVECs, of which 10 were upregulated and 21 were downregulated. qRT-PCR and ELISA studies were in accordance with the array. DMVECs expressed a mixed profile of factors that can contribute to and inhibit scar formation. HTS DMVECs have both a discordant response to cellular insults and baseline differences in function, supporting their proposed role in scar pathology. Further investigation of DMVECs is warranted to elucidate their contribution to HTS pathogenesis.

Comparison of the Efficiency of Magnetic-Activated Cell Sorting (MACS) and Physiological Intracytoplasmic Sperm Injection (PICSI) for Sperm Selection in Cases with Unexplained Infertility.

The cases with unexplained infertility may have an abnormality in their sperm chromatin structure. Sperm selection methods can be used to separate sperm with low DNA fragmentation. The purpose of this study was to compare the efficacy of physiological intracytoplasmic sperm injection (PICSI) with magnetic-activated cell sorting (MACS) in assisted reproductive techniques in cases with unexplained infertility. The semen samples were collected from couples with unexplained infertility. After semen analysis and sperm DNA fragmentation (SDF) evaluations, samples were prepared with swim-up method. The rates of SDF in different fractions including raw semen (n=20), swim-up (n=20), only motile sperm after swim-up (swim-up selection) (n=20), MACS sperm selection (n=20), only motile sperm after MACS (MACS selection) (n=20), and PICSI sperm selection (n=16) were evaluated. Also, the main sperm characteristics and fine morphology of sperm suspension after MACS were assessed. Statistical analysis was performed using GraphPad Prism. The p<0.05 was considered statistically significant. DNA fragmentation index (DFI) values in PICSI and MACS groups were significantly reduced as compared to the swim-up group. The rate of this reduction was more pronounced in MACS (58.20±13.02) than PICSI (36.57±15.52) group. Also, our results showed that MACS resulted in decreased sperm motility, with no alteration in their fine morphology. MACS was found to be more efficient in reduction of SDF rates than PICSI. However, none of the sperm selection techniques can not totally eliminated the spermatozoa with DNA fragmentation in the final sperm sample.

Photothermal effect of albumin-modified gold nanorods diminished neuroblastoma cancer stem cells dynamic growth by modulating autophagy

Here, we investigated the photothermal effect of gold nanorods (GNRs) on human neuroblastoma CD133+ cancer stem cells (CSCs) via autophagic cell death. GNRs were synthesized using Cetyltrimethylammonium bromide (CTAB), covered with bovine serum albumin (BSA). CD133+ CSCs were enriched from human neuroblastoma using the magnetic-activated cell sorting (MACS) technique. Cells were incubated with GNRs coated with BSA and exposed to 808-nm near-infrared laser irradiation for 8 min to yield low (43 °C), medium (46 °C), and high (49 °C) temperatures. After 24 h, the survival rate and the percent of apoptotic and necrotic CSCs were measured using MTT assay and flow cytometry. The expression of different autophagy-related genes was measured using polymerase chain reaction (PCR) array analysis. Protein levels of P62 and LC3 were detected using an enzyme-linked immunosorbent assay (ELISA). The viability of CSC was reduced in GNR-exposed cells compared to the control group (p < 0.05). At higher temperatures (49 °C), the percent of apoptotic CSCs, but not necrotic cells, increased compared to the lower temperatures. Levels of intracellular LC3 and P62 were reduced and increased respectively when the temperature increased to 49 °C (p < 0.05). These effects were non-significant at low and medium temperatures (43 and 46 °C) related to the control CSCs (p > 0.05). The clonogenic capacity of CSC was also inhibited after photothermal therapy (p < 0.05). Despite these changes, no statistically significant differences were found in terms of CSC colony number at different temperatures regardless of the presence or absence of HCQ. Based on the data, the combination of photothermal therapy with HCQ at 49 °C can significantly abort the CSC clonogenic capacity compared to the control-matched group without HCQ (p < 0.0001). PCR array showed photothermal modulation of CSCs led to alteration of autophagy-related genes and promotion of co-regulator of apoptosis and autophagy signaling pathways. Factors related to autophagic vacuole formation and intracellular transport were significantly induced at a temperature of 49 °C (p < 0.05). We also note the expression of common genes belonging to autophagy and apoptosis signaling pathways at higher temperatures. Data showed tumoricidal effects of laser-irradiated GNRs by the alteration of autophagic response and apoptosis.

Isolation of Leydig cells from adult rat testes by magnetic-activated cell sorting protocol based on prolactin receptor expression.

The primary function of testicular Leydig cells (LCs) is to produce testosterone (T). In vitro culture of the cells represents a very important approach to study androgen production and its regulations. Various methods have been developed for the enrichment of the cells from the testes. However, getting cells in large numbers with high purity and viability is still challenging. Here, we describe a new way to isolate LCs from rat testes in large quantity with high purity and viability. Enzymatic digested testicular cells from adult rats were labelled with prolactin receptor (PRLR) antibody. The positive cells were isolated by magnetic-activated cell sorting (MACS) protocol. Purified LCs were tested in vitro for their steroidogenic (T production) and non-steroidogenic (25-OH-vitamin D production and Insl3 and Cyp2r1expressions) functions in the presence of Luteinizing Hormone (LH) for up to 24h. Reanalysis of scRNA-seq data indicates that Prlr expression is highly specific in LCs of adult rat testis. MACS procedure based on PRLR expression was able to isolate LCs with very high yield (about 106 cells/testis), high purity (about 95%) and viability (>93%). Purified LCs retained high steroidogenic and non-steroidogenic functions in responding to maximal LH stimulations, with more than 10-fold increases in T production in 3h and 42% and 103% increases in Insl3 and Cyp2r1 expressions in 24h. We have established an excellent way to purify high quality LCs from adult rat testis that can serve as a useful tool to study the physiology, pharmacology and toxicology of the cells in vitro.

P-006 MACS Vs Microfluidics Sperm Sorting FOR RAISED SPERM DNA FRAGMENTATION INDEX – A RCT

Abstract Study question In Individuals with raised Sperm DNA Fragmentation Index (DFI), will sperm selection by magnetic activated cell sorting (MACS) or Microfluidic Sperm Sorting (MF) optimize the reproductive outcomes? Summary answer MACS or MF offer comparable outcomes. There seems no superiority between them. Individuals with raised spermDFI, MACS/MF can be considered to optimize results. What is known already It is evident that raised sperm DFI negatively affects the reproductive outcomes. Management for raised sperm DFI to optimize reproductive outcomes is still elusive. Study design, size, duration This is an ongoing Randomized Control Trial (RCT) with prior approval from institutional Ethical Committee (IEC) (ECR/1312/Inst/TG/2019). This is preliminary data of this ongoing study. Study duration 6months(July 2020–January 2021). Couples undergoing IVF stimulation with raised spermDFI were randomized into MACS(n = 75) and MF(n = 58) for sperm selection. This study also intends to look at efficiency of MACS and MF sperm sorting to reduce spermDFI post intervention. This trial is registered with Clinical Trials Registry India (CTRI) REF/2021/05/043439 Participants/materials, setting, methods Couples with one failed IVF were offered sperm DFI. Individuals with sperm DFI&amp;gt;30% were recruited. Sperm DFI testing was done with SCSA method and randomized using software. Sperm processing done with MACS or MF based on randomization on the day of Oocyte Retrieval(OPU). MACS/MF sperm processing done as per manufacturer’s instructions. Culture done till blastocyst stage and freeze all policy followed. Two Blastocysts that showed100% survival was transferred in a Frozen Embryo transfer (FET) cycle. Main results and the role of chance Implantation rates(IR) Miscarriage Rates (MR), Multiple Pregnancy Rates(MPR) Live Birth rates (LBR) were compared between groups Reduction in sperm DFI value between MACS and MF was also evaluated In MACS arm - 75 subjects recruited, 49 patients (65%) had atleast 2 blastocysts frozen and underwent FET. 26 patients (35%) had cancelled cycles due to no blastocyst formation. In MF arm - 58 subjects recruited 38 patients (65%) had atleast 2 blastocysts and underwent FET. 20 patients (35%) had cancelled cycles due to no blastocyst formation. Sperm DFI reduction with MACS Vs MF Sperm Sorting 83% of men had sperm DFI value reduced to &amp;lt; 30% post MACS intervention 87% of men had sperm DFI value reduced to &amp;lt; 30% post MF intervention Both MACS and MF seem to be efficient in sorting sperm with raised DFI and separate sperms with good DNA quality Reproductive Outcomes of MACS Vs MF Sperm Sorting were IR - 50% Vs 48% (p = 0.8540) MR - 5.3% Vs 11% (p = 0.3280) MPR - 8% Vs 18% (p = 0.1624) LBR per ET cycle - 63% Vs 63% (p = 1.0000) Reproductive Outcomes of MACS and MF sperm Sorting seems to be comparable. Data from this study doesn’t prove superiority of one processing method over another Limitations, reasons for caution This is an ongoing study and the data presented here is the preliminary data with small sample size Wider implications of the findings Optimal intervention for management of raised spermDFI needs further research. However, MACS/MF both are non invasive and seem to optimize reproductive outcomes. Nevertheless while offering these sperm sorting methods aspects like cost, time taken to process and technical challenges if any, should be considered and offered with appropriate counselling. Trial registration number REF/2021/05/043439

P-008 Microgravity exposure significantly decreases sperm motility and vitality. Can we consider human reproduction outside the Earth?

Abstract Study question Are fresh human sperm samples affected by different gravitational conditions than on Earth? Summary answer Motility and vitality of fresh human sperm samples are significantly decreased under microgravity conditions obtained by parabolic flight. What is known already Microgravity effects on the male reproductive system have mainly been studied in the animal model with diverse results and discouraging extrapolation in humans. While an increased motility was reported in bulls, mice models showed a decrease. Although preliminary data from the Micro-11 experiment presented by NASA reported human sperm alterations after microgravity exposure, our first study failed to show any significant effect of microgravity on “frozen” samples, suggesting that human sperm could be safely shipped outside the earth if important aspects related with cryopreservation were solved. Study design, size, duration Prospective study carried out in collaboration between the ART centre, a Technical University, and an Aviation Club specializing in parabolic flights.Two parabolic flights were conducted between 2020-2021, each consisting of 20 parabolic maneuvers, which means 160 seconds of microgravity exposure per sample. Fifteen sperm samples obtained from healthy men were included in the study in order to analyse the effects of microgravity and compare the results with those obtained in Earth gravity. Participants/materials, setting, methods Fresh sperm samples were checked pre-flight to evaluate vitality, concentration, motility and morphology. Samples were split into two to compare the effects of different gravity exposure: microgravity (flight) and Earth gravity (ground). After the flight, the same analysis were repeated, plus kinematics, DNA fragmentation by sperm chromatin dispersion, apoptosis by magnetic activated cell sorting, and oxidative stress by colorimetric test (Halosperm-Halotech). Computer Aided Semen Analysis (SCA-Scope) was used for cell counting. Main results and the role of chance On comparison of the mean values between fresh samples exposed to microgravity and those maintained on Earth gravity, statistical significant differences (p &amp;lt; 0,05) were found in the following parameters: vitality (69,7 ± 9,9 vs 72,4 ± 9,7 %), motile sperm concentration (23,7 ± 15,3 M/ml vs 31,5 ± 25,1 M/ml), grade “a” sperm concentration (8,7 ± 6,5 vs 11,7 ± 9,9 M/ml), percentage of spermatozoa with progressive motility (30 ± 12,9 vs 36 ± 14,3 %), curvilinear motility-VCL (45,7 ± 12,8 vs 47,7 ± 13,3 μm/s). Under the study conditions, non-statistically significant differences were observed in the other kinematic parameters: Lineal Velocity (VSL), Average Path Velocity (VAP), Straightness (STR), Amplitude of Lateral Head displacement (ALH), Linearity (LIN), Wobble (WOB), Beat-Cross Frequency (BCF), total sperm concentration (81,7 ± 112,1 vs 79,7 ± 89,8 M/ml), morphology (11,3 ± 6,3 vs 10,6 ± 5,3%), DNA fragmentation (14,6 ± 9,6 vs 15,7 ± 9,4), apoptosis (2,8 ± 2,8 vs 3,8 ± 4,4) and oxidative stress, since all samples maintained the same stress level in both splits. Limitations, reasons for caution Parabolic flight is an accepted ground-based method for obtaining microgravity conditions, but provides a short period of elapsed exposure to microgravity. Therefore, the results obtained need to be confirmed by using other platforms that provide a much longer time of exposure. More cases must be analysed to confirm the results. Wider implications of the findings Short exposure to microgravity significantly decreases sperm motility and vitality. Such an effect is likely to be stronger with longer exposure. These findings should be taken into account since this may eventually affect sperm fertilizing capacity and therefore natural conception or ART with fresh/frozen sperm, outside of the Earth. Trial registration number NCT03760783

Cav1.2 regulated odontogenic differentiation of NG2+ pericytes during pulp injury.

NG2+ pericytes, as the possible precursor cells of mesenchymal stem cells (MSCs), have drawn attention due to their ability to differentiate into odontoblasts. Cav1.2 is involved in the differentiation process of stem cells, but its role in the differentiation of NG2+ pericytes is not clear. The aim of the present study was to examine the role of Cav1.2 in the differentiation of NG2+ pericytes into odontoblasts. NG2+ pericytes were obtained from human dental pulp cells by magnetic-activated cell sorting. During the odontogenic differentiation of NG2+ pericytes, the effects of the Cav1.2 inhibitors, nimodipine and Cav1.2 knockdown shRNA, were analyzed by real-time polymerase chain reaction and alizarin red staining. NG2CreERT2/Rosa26-GFP lineage-tracing mice were established to further investigate the roles of NG2+ pericytes and Cav1.2 in incisor self-repair after injury in vivo. At 10min, 1day, and 3days after pulp injuries in transgenic mice, NG2-GFP+ and Cav1.2 immunofluorescence co-staining was performed on the incisors. Nimodipine treatment and Cav1.2 knockdown showed similar inhibition of calcium nodule formation and mRNA levels of osteogenic markers (DSPP, DMP1, and Runx2, p < 0.05). NG2+ pericytes migrated from their inherent perivascular location to the odontoblast layers after pulp injury. Cav1.2 showed a similar response pattern as NG2+ pericytes and gradually returned to normal levels. In addition, many co-stained areas of Cav1.2 and NG2+ pericytes, both near the perivascular and odontoblast layers, were observed. These results indicate that Cav1.2 played a vital role in the odontogenic differentiation of NG2+ pericytes, and that it might be closely linked to the NG2+ pericytes-mediated repair of dental pulp injury in vivo.

Human primary skeletal muscle‐derived myoblasts and fibroblasts reveal different senescent phenotypes

AbstractBackgroundThe age‐related loss of muscle mass and quality, sarcopenia, has many contributing factors, one of which may be cellular senescence, but this is not well defined in human skeletal muscle.MethodPrimary cells were isolated from biopsy samples of the vastus lateralis muscle from healthy adult males (n = 6, 22 ± 1 years), sorted (magnetic activated cell sorting) and chemically induced (doxorubicin, DOX, 0.2 μM) to a senescent state. This allowed the parallel and simultaneous investigation of the two main skeletal muscle‐derived cell types: satellite cell‐derived CD56+ve/desmin+ve myoblasts (muscle precursor cells) and CD56− ve/TE7+ve fibroblasts (at &gt;95% purity). Both cell types were followed for up to 35 days post DOX treatment with a combination of quantitative immunocytochemistry and qRT‐PCR for senescent markers and senescence‐associated secretory phenotype (SASP) factors.ResultsMyoblasts and fibroblasts showed temporal and quantitative differences in many of the senescence markers studied. p16 protein expression increased across the time course (P &lt; 0.0001) with no difference between cell types, whereas at the mRNA level, myoblasts showed increased p16 expression from 4 days post treatment (FC = 3.03 ± 0.99), and in fibroblasts, this appeared later at 10 and 35 days post DOX treatment (FC = 8.09 ± 2.46, P &lt; 0.0001). Both myoblasts (FC = 8.83 ± 1.72) and fibroblasts (FC = 2.33 ± 1.10) showed significant increases in p21 mRNA (P &lt; 0.0001), which remained elevated in the myoblast cell populations across the 35‐day time course but returned to baseline in the fibroblasts from 4 days post DOX treatment. Within 35 days post DOX treatment, all cell populations of both myoblasts and fibroblasts had reached 100% SA‐β‐Gal‐positive cells (P &lt; 0.05). γH2aX expression (a marker of DNA damage) increased 1 day after DOX treatment in the myoblasts (FC = 3.3 ± 1.1, P &lt; 0.05) but returned to baseline within 4 days post DOX treatment, whereas fibroblasts showed a similar trend that did not reach statistical significance. Significant reductions in expression of the proliferation marker Ki67 1 day post DOX treatment were seen in both cell types and were maintained throughout the time course (FC = 0.11 ± 0.07, P &lt; 0.0001). Significant changes over the time course were also observed in mRNA expression of selected SASP factors (e.g. PAI‐1, MMP3, and IGFBP3, P &lt; 0.05).ConclusionsNeither cellular senescence nor sarcopenia is fully understood. The present data on human primary myoblasts and fibroblasts obtained from the same tissue sample show that senescence is a complex, non‐linear, and dynamic cellular process which shows intra‐ and inter‐cell variability.

Purification of human iPSC-derived cells at large scale using microRNA switch and magnetic-activated cell sorting.

SummaryFor regenerative cell therapies using pluripotent stem cell (PSC)-derived cells, large quantities of purified cells are required. Magnetic-activated cell sorting (MACS) is a powerful approach to collect target antigen-positive cells; however, it remains a challenge to purify various cell types efficiently at large scale without using antibodies specific to the desired cell type. Here we develop a technology that combines microRNA (miRNA)-responsive mRNA switch (miR-switch) with MACS (miR-switch-MACS) to purify large amounts of PSC-derived cells rapidly and effectively. We designed miR-switches that detect specific miRNAs expressed in target cells and controlled the translation of a CD4-coding transgene as a selection marker for MACS. For the large-scale purification of induced PSC-derived cardiomyocytes (iPSC-CMs), we transferred miR-208a-CD4 switch-MACS and obtained purified iPSC-CMs efficiently. Moreover, miR-375-CD4 switch-MACS highly purified pancreatic insulin-producing cells and their progenitors expressing Chromogranin A. Overall, the miR-switch-MACS method can efficiently purify target PSC-derived cells for cell replacement therapy.

The synergistic anticancer effect of salinomycin combined with cabazitaxel in CD44+ prostate cancer cells by downregulating wnt, NF-κB and AKT signaling.

Tumor-initiating or cancer stem cells (CSCs) reduce the effectiveness of conventional therapy. Thus, it is crucial to eliminate CSCs while killing bulky cancer cells using a combination of conventional chemotherapy and anti-CSC drugs. Salinomycin is a selective inhibitor against CSCs and shows promise in combination applications. The aim of the study was to examine the efficacy of co-administered cabazitaxel and salinomycin on the survival of prostate cancer cells and CSCs. CD44 + stem cells were isolated from human PC3 prostate cancer cells by using magnetic activated cell sorting. The cells were concomitantly exposed to salinomycin and cabazitaxel, and the cell survival was determined by MTT test. Apoptosis was assessed by image-based cytometer, and cell migration was evaluated by wound healing assay. The expression of target mRNA and protein were assessed by RT-qPCR and Western blot, respectively. Combination index (CI) analysis showed that simultaneous administration of salinomycin and cabazitaxel was able to exert strong synergistic effect on CD44 + subpopulation (CI = 0.33), but no synergism was observed in PC3 cells. The combination of the two agents significantly increased Bax, cytochrome c, caspase-3 and - 8 mRNA expression in CD44 + CSCs, causing apoptosis. The applied therapy strategy strongly inhibited the phosphorylation of Akt, protein expression of Akt1, NF-κB and Wnt. In conclusion, our data suggest that combining salinomycin with cabazitaxel shows promise as a prostate cancer treatment approach that can target CSCs.

Effect of Sperm Selection by Magnetic-Activated Cell Sorting in D-IUI: A Randomized Control Trial.

Clinical outcome in assisted reproduction techniques (ARTs) is mainly influenced by the quality of gametes used. It is known that a high percentage of sperm DNA fragmentation (DNAf) decreases the success of ART clinical results. Therefore, techniques such as magnetic-activated cell sorting (MACS) help to improve results in cases of patients with a high percentage of DNAf. Cryopreservation of sperm in donor intrauterine insemination (D-IUI) treatments increases sperm DNAf, so patients using these sperm samples can benefit from using this technique. This prospective randomized national multicenter study analyzed clinical outcomes of 181 D-IUI treatments. MACS was performed after density gradient centrifugation (DGC) in 90 thawed semen donor samples (MACSG), whereas only DGC was performed in 91 thawed semen donor samples (CG). To our knowledge, this is the first study analyzing the effect of MACS on D-IUI cycles. Our results show no significant differences in gestation, live birth, or miscarriage rates between the two groups. We believe that further studies with a larger sample size are needed to evaluate the application of MACS in combination with standard IUI donor sperm preparations in fertility clinics.

Patients With Inflammatory Bowel Disease Show IgG Immune Responses Towards Specific Intestinal Bacterial Genera.

IntroductionInflammatory bowel disease (IBD) is characterized by a disturbed gut microbiota composition. Patients with IBD have both elevated mucosal and serum levels of IgG-antibodies directed against bacterial antigens, including flagellins. In this study, we aimed to determine to which intestinal bacteria the humoral immune response is directed to in patients with IBD.MethodsFecal and serum samples were collected from patients with IBD (n=55) and age- and sex-matched healthy controls (n=55). Fecal samples were incubated with autologous serum and IgG-coated fractions were isolated by magnetic-activated cell sorting (MACS) and its efficiency was assessed by flow cytometry. The bacterial composition of both untreated and IgG-coated fecal samples was determined by 16S rRNA-gene Illumina sequencing.ResultsIgG-coated fecal samples were characterized by significantly lower microbial diversity compared to the fecal microbiome. Both in patients with IBD and controls, serum IgG responses were primarily directed to Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Veillonella and Enterobacteriaceae, as well as against specific Lachnospiraceae bacteria, including Coprococcus and Dorea (all P<0.001), and to Ruminococcus gnavus-like bacteria (P<0.05). In contrast, serological IgG responses against typical commensal, anaerobic and colonic microbial species were rather low, e.g. to the Lachnospiraceae members Roseburia and Blautia, to Faecalibacterium, as well as to Bacteroides. Patients with IBD showed more IgG-coating of Streptococcus, Lactobacillus, and Lactococcus bacteria compared to healthy controls (all P<0.05). No differences in IgG-coated bacterial fractions were observed between Crohn’s disease and ulcerative colitis, between active or non-active disease, nor between different disease locations.ConclusionThe IgG immune response is specifically targeted at distinct intestinal bacterial genera that are typically associated with the small intestinal microbiota, whereas responses against more colonic-type commensals are lower, which was particularly the case for patients with IBD. These findings may be indicative of a strong immunological exposure to potentially pathogenic intestinal bacteria in concordance with relative immune tolerance against commensal bacteria.

Magnetic-Activated Cell Sorting as a Method to Improve Necrozoospermia-Related Asthenozoospermic Samples.

According to some statistics, absolute asthenozoospermia affects every 1 in 5000 men. Although this incidence rate does not appear to be too high, it is extremely important to address the phenomenon because it can drastically reduce the chances of pregnancy, even with assisted reproduction. The biggest problem with absolute asthenozoospermia is that it is difficult to distinguish between live and dead sperm cells, and fertilization with non-viable spermatozoa may contribute to the failure of an assisted reproduction cycle. Nowadays, DNA fragmentation (DF) is a crucial parameter of semen analysis, and in this paper, we provide evidence of the correlation between DF and vitality. For this purpose, the main semen parameters were investigated by a CASA system (concentration, motility, progressive motility, vitality and DF). In the necrozoospermic group (vitality < 58%), all the measured parameters showed significant differences compared to normal vitality. Concentration (30.1 M mL−1 vs. 13.6 M mL−1), motility (31.9% vs. 18.3%), and progressive motility (24.3% vs. 12.7%) were significantly decreased, while DF was significantly increased (17.4% vs. 23.7%). Based on the connection between vitality decrement and DF increment, DF lowering methods, such as magnetic-activated cell sorting, have been hypothesized as novel methods for the elimination of dead spermatozoa.

High-Efficiency Bovine Sperm Sexing Used Magnetic-Activated Cell Sorting by Coupling scFv Antibodies Specific to Y-Chromosome-Bearing Sperm on Magnetic Microbeads

Simple SummaryFemale calves are favored for milk production and genetic advancement in the dairy industry, and sex selection by using sexed semen has been long considered. A potential alternative sperm sexing technique is magnetic-activated cell sorting combined with an immunological method that uses scFv antibodies against male-specific sites on Y-chromosome-bearing sperm; however, the technique should be evaluated for validity and accuracy. This study focuses on how well bovine sperm are separated by the use of magnetic microbeads coupled with scFv antibodies against Y-chromosome-bearing sperm (PY-microbeads). The results showed that sexed bovine sperm using PY-microbeads was a highly effective technique for distinguishing X- and Y-chromosome-bearing sperm. It had no negative impact on the quality of X-chromosome-bearing sperm. The technique produced 82.65% of X-chromosome sperm in the X-enriched fraction semen and 81.43% of Y-chromosome sperm in the Y-enriched fraction semen, which was utilized to generate target sexed bovine semen.Sperm sexing technique is favored in the dairy industry. This research focuses on the efficiency of bovine sperm sexing using magnetic-activated cell sorting (MACS) by scFv antibody against Y-chromosome-bearing sperm (Y-scFv) coupled to magnetic microbeads and its effects on kinematic variables, sperm quality, and X/Y-sperm ratio. In this study, the optimal concentration of Y-scFv antibody coupling to the surface of magnetic microbeads was 2–4 mg/mL. PY-microbeads revealed significantly enriched Y-chromosome-bearing sperm (Y-sperm) in the eluted fraction (78.01–81.43%) and X-chromosome-bearing sperm (X-sperm) in the supernatant fraction (79.04–82.65%). The quality of frozen–thawed sexed sperm was analyzed by CASA and imaging flow cytometer, which showed that PY-microbeads did not have a negative effect on X-sperm motility, viability, or acrosome integrity. However, sexed Y-sperm had significantly decreased motility and viability. The X/Y-sperm ratio was determined using an imaging flow cytometer and real-time PCR. PY-microbeads produced sperm with up to 82.65% X-sperm in the X-enriched fraction and up to 81.43% Y-sperm in the Y-enriched fraction. Bovine sperm sexing by PY-microbeads showed high efficiency in separating Y-sperm from X-sperm and acceptable sperm quality. This initial technique is feasible for bovine sperm sexing, which increases the number of heifers in dairy herds while lowering production expenses.

Functional Characterization of Endothelial Cells Differentiated from Porcine Epiblast Stem Cells.

Endothelial cells (ECs), lining blood vessels’ lumen, play an essential role in regulating vascular functions. As multifunctional components of vascular structures, pluripotent stem cells (PSCs) are the promising source for potential therapeutic applications in various vascular diseases. Our laboratory has previously established an approach for differentiating porcine epiblast stem cells (pEpiSCs) into ECs, representing an alternative and potentially superior cell source. However, the condition of pEpiSCs-derived ECs growth has yet to be determined, and whether pEpiSCs differentiate into functional ECs remained unclear. Changes in morphology, proliferation and functional endothelial marker were assessed in pEpiSCs-derived ECs in vitro. pEpiSCs-derived ECs were subjected to magnetic-activated cell sorting (MACS) to collect CD-31+ of ECs. We found that sorted ECs showed the highest proliferation rate in differentiation media in primary culture and M199 media in the subculture. Next, sorted ECs were examined for their ability to act as typical vascular ECs through capillary-like structure formation assay, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake, and three-dimensional spheroid sprouting. Consequently, pEpiSCs-derived ECs function as typical vascular ECs, indicating that pEpiSC-derived ECs might be used to develop cell therapeutics for vascular disease.