Magnetic-activated Cell Sorting Technique Research Articles

Immunomodulatory effects of long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) on porcine monocytes (CD14 +) immune response in vitro

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 long-chain polyunsaturated fatty acids (n-3 PUFA) found mostly in fish oil. They have been commonly used as dietary integrators in human and animal nutrition, modulating the immune system, mostly by exerting anti-inflammatory activities as demonstrated by in vivo and in vitro studies. The precise mechanisms of action at the background of EPA and DHA immunomodulatory activity are still not fully elucidated. Moreover, no information on their effects on porcine monocytes immune response is available yet. To cover this gap, the study aimed to evaluate DHA and EPA's in vitro impact on porcine monocytes (CD14 +) defensive functions. Briefly, monocytes were isolated from the blood of twenty-six healthy pigs, using a magnetic-activated cell sorting technique (MACS). Monocytes were first treated with increasing concentrations of DHA and EPA (25, 50, 100 and 200 µM) and apoptosis and viability were measured to assess potential cytotoxic effects. Once determined EPA and DHA subtoxic working concentrations (25, 50 and 100 µM), their effects on chemotaxis, phagocytosis and total, intracellular and extracellular reactive oxygen species (ROS) production were evaluated. DHA and EPA only decreased porcine monocytes viability at the highest concentration (200 µM), but their apoptosis was unaffected. DHA (100 µM) decreased the cells’ chemotaxis, while EPA (25 µM) increased their intracellular ROS production after 60 min under non-inflammatory or resting conditions and at 90 min under pro-inflammatory conditions (PMA challenge). EPA (50 µM) decreased monocytes’ intracellular ROS levels only under resting conditions at 30 min. No effects were observed on porcine monocytes phagocytic capacity. In conclusion, this study demonstrates that DHA and EPA can exert differential in vitro immunomodulatory effects in pigs, by dampening monocytes chemotaxis and potentiating their oxidative burst, respectively. Thus, our results suggest these n-3 PUFA might exert both anti-inflammatory and/or immune-enhancing effects in pigs.

The potential benefits of dinitrophenol combination with chemotherapy in the treatment of ovarian cancer.

2,4-dinitrophenol (DNP), an uncoupling mitochondrial agent, has been identified as a source of oxidative stress and linked to the pathogenesis of ovarian cancer. In this study, we determine the cytotoxic effect of DNP alone or in combination with chemotherapies in ovarian cancer cells. We utilized human ovarian cancer cell lines SKOV-3 and MDAH-2774 with their chemoresistant counterparts. Cancer stem cells (CSCs) were isolated from SKOV-3 utilizing magnetic-activated cell sorting technique for CD44+/CD117+ cells. Human normal primary ovarian epithelial (NOEC) and HOSEpiC cell lines were used as a control. Cells were treated with and without chemotherapy (Taxotere 0.3μM or cisplatin 50 μM), with or without increasing doses of DNP (0.125, 0.25, or 0.5 mM) for 24 hours followed by evaluation of cell viability and IC50 utilizing MTT assay. For determination of synergism, Facombination index plots were created using the CompuSyn software. All data were run in triplicates and analyzed by t-test. DNP treatment of ovarian cancer and chemoresistant ovarian cancer cell lines as well as CSCs resulted in decreased cell viability in a dose dependent manner with no effect on normal cells. Combination of DNP with chemotherapy synergistically enhances cytotoxicity of chemotherapeutics in all ovarian cancer cells as compared to chemotherapy alone. Our data indicates the potential of the addition of DNP to the arsenal of drugs available to treat ovarian cancer, whether alone or in combination with chemotherapies. The synergistic effects of DNP in reducing the required amount of chemotherapy, is critical for the alleviation of harmful side effects.

Effects of Midazolam on the Development of Adult Leydig Cells From Stem Cells In Vitro.

BackgroundMidazolam is a neurological drug with diverse functions, including sedation, hypnosis, decreased anxiety, anterograde amnesia, brain-mediated muscle relaxation, and anticonvulsant activity. Since it is frequently used in children and adolescents for extended periods of time, there is a risk that it may affect their pubertal development. Here, we report a potential effect of the drug on the development of Leydig cells (LCs), the testosterone (T)-producing cells in the testis.MethodsStem LCs (SLCs), isolated from adult rat testes by a magnetic-activated cell sorting technique, were induced to differentiate into LCs in vitro for 3 weeks. Midazolam (0.1–30 μM) was added to the culture medium, and the effects on LC development were assayed.ResultsMidazolam has dose-dependent effects on SLC differentiation. At low concentrations (0.1–5 μM), the drug can mildly increase SLC differentiation (increased T production), while at higher concentrations (15–30 μM), it inhibits LC development (decreased T production). T increases at lower levels may be due to upregulations of scavenger receptor class b Member 1 (SCARB1) and cytochrome P450 17A1 (CYP17A1), while T reductions at higher levels of midazolam could be due to changes in multiple steroidogenic proteins. The uneven changes in steroidogenic pathway proteins, especially reductions in CYP17A1 at high midazolam levels, also result in an accumulation of progesterone. In addition to changes in T, increases in progesterone could have additional impacts on male reproduction. The loss in steroidogenic proteins at high midazolam levels may be mediated in part by the inactivation of protein kinase B/cAMP response element-binding protein (AKT/CREB) signaling pathway.ConclusionMidazolam has the potential to affect adult Leydig cell (ALC) development at concentrations comparable with the blood serum levels in human patients. Further studies are needed to test the effects on human cells.

8 Effects of magnetic-activated cell sorting on human sperm motility and DNA fragmentation index

Selection of spermatozoa before their use for assisted reproductive techniques is an important step in therapy of human infertility. The DNA fragmentation index of sperm plays a major role in pregnancy rates following IVF and intracytoplasmic sperm injection (ICSI). Sperm analyses and standard sperm selection methods in many cases do not eliminate a sufficient proportion of sperm with apoptosis and DNA fragmentation. Magnetic-activated cell sorting (MACS) is a selection method that eliminates apoptotic spermatozoa based on the presence of externalized phosphatidylserine residues. The aim of our study was to evaluate the effect of MACS on human sperm motility and DNA fragmentation index (DFI) in a patient population. The participants were 63 male patients of an IVF clinic, 34 to 45 years old, with 3 years of primary infertility due to male factor. Semen analysis was performed according to the World Health Organization guidelines (2010) and revealed oligoasthenoteratozoospermia in 63 patients. The DFI of fresh semen samples was evaluated using the sperm chromatin structure assay (SCSA) test and revealed DFI 32.4±5.9%. The SCSA test was done on a flow cytometer CyFlow Space (Sysmex-Partec; Evenson 2016 Anim. Reprod. Sci. 169, 56-75; https://doi.org/10.1016/j.anireprosci.2016.01.017). Sperm motility was studied on Hamilton Thorne IVOS. For MACS, we used the MACS® ART Annexin V system (Miltenyi Biotec). The semen sample was diluted to a concentration 10×106 spermatozoa mL−1. After double-density gradient centrifugation, the pellet was resuspended in 100µL of MACS Art Annexin V reagent and added MACS Art Binding Buffer (BB) to 500µL. The sample was gently mixed and incubated for 15min at room temperature. After rinsing the column with BB, the sperm-bead suspension was added on top with BB and, immediately after that, the annexin V-negative and annexin V-positive fractions were obtained (MiniMACS; Miltenyi Biotec). Data were evaluated by ANOVA Student’s t-test. Fresh semen samples collected from the patients had an average sperm concentration of 29.7±5.7×106 mL−1, motility of 32.7±5.9%, and DFI of 32.4±5.9%. Motility of spermatozoa after MACS for the annexin-negative fraction was 47.2±6.3% and for the annexin-positive fraction was 3.5±2.3% (P<0.003). Similarly, the annexin-negative spermatozoa had a lower DFI (10.5±3.8%) rate than did the annexin-positive fraction (67.8±5.9%; P<0.003). The MACS technique allowed a significant reduction of DNA fragmentation levels (from 32.4% for the original sample to 10.5% for the annexin-negative; P<0.01). The separation of a distinct population of nonapoptotic spermatozoa with intact membranes may optimize outcomes from IVF and ICSI procedures. Magnetic-activated cell sorting of human sperm using annexin-V microbeads results in selection of a population with enhanced motility and reduced DFI rates.

Magnetic-Activated Cell Sorting (MACS): A Useful Sperm-Selection Technique in Cases of High Levels of Sperm DNA Fragmentation

Magnetic-activated cell sorting (MACS) can be used to separate apoptotic sperm with high proportions of fragmented DNA from the rest, thus improving the overall quality of the seminal sample. Therefore, the aim of this retrospective study was to investigate the efficiency of the MACS technique to increase reproductive outcomes in patients with high levels of sperm DNA fragmentation (SDF) undergoing intracytoplasmic sperm-injection (ICSI) cycles. In this study, we analyzed a total of 724 assisted-reproduction-technique (ART) cycles that were divided into two groups: the study group (n = 366) in which the MACS selection technique was performed after density-gradient centrifugation (DGC), and the control group (n = 358) in which only DGC was used for sperm selection. Reproductive outcomes were analyzed in both groups according to three different ART procedures: preimplantation genetic testing for aneuploidy (PGT-A), and autologous and oocyte-donation cycles. The MACS group showed significantly lower miscarriage rates in autologous ICSI cycles, higher pregnancy rates in oocyte-donation cycles, and a significant increase in live-birth rates in both autologous and oocyte-donation cycles. Overall, these results suggested that the MACS technique can be effectively used to eliminate sperm with high SDF levels, and therefore may help to improve reproductive outcomes in couples undergoing ART.

Healthy baby born after ICSI with ejaculated immotile spermatozoa from a male Kartagener syndrome using Magnetic-Activated Cell Sorting (MACS) as a compliment of sperm preparation technique: A case report

Kartagener syndrome is an autosomal recessive genetic disorder associated with male infertility. Mutations in genes that encode a protein called dynein affect sperm motility.A good option for these patients is to enhance the sperm selection for ICSI that could lead to improve the reproductive outcomes. For this purpose, different strategies have been used successfully in isolated cases.In the present case report, we successfully applied MACS technique to a semen sample. This immunomagnetic method of sperm selection was aimed at reducing apoptosis manifestations, including DNA fragmentation, to optimize the outcomes of the ICSI procedure. By doing so, we increased the chances of selecting spermatozoa with superior quality and higher fertilization potential in the presence of total asthenozoospermia, even after incubation with pentoxifylline. Six of ten oocytes (60%) were appropriately fertilized. Two good-quality embryos were transferred on day 3 resulting in a pregnancy and a healthy baby born and one cavitated blastocyst was frozen on day 5.MACS technique used as a compliment of sperm preparation technique may be a promising approach in routine IVF practice for men with Kartagener syndrome to improve reproductive outcomes optimizing the outcomes of the ICSI procedure.

Seminal plasma and CD4+ T-cell cytokine profiles in the in vitro fertilization success.

Background:Abnormal female immune response is one of the potential causes of unexplained infertility (UI). Seminal plasma (SP) is an important regulator of female immune responses during pregnancy. This study investigated a SP effect on the expression of CD4+ T-cell-related cytokines in a group of UI woman candidates for in vitro fertilization (IVF) and healthy fertile women.Materials and Methods:This was a semi-experimental study that performed on 20 UI couples (ten unsuccessful and ten successful IVF outcomes) and 10 fertile couples as the healthy group. CD4+ T-cells were separated from peripheral blood mononuclear cells of women by magnetic-activated cell sorting technique and incubated with (stimulated condition) or without (unstimulated condition) SP of their husbands. After incubation, real-time polymerase chain reaction method was used to investigate interleukin (IL)-23, IL-17, IL-4, IL-10, transforming growth factor (TGF)-β, and interferon (IFN)-γ gene expression. Mann–Whitney U-test, Kruskal–Wallis test, and Wilcoxon signed-rank test were used for statistical analysis.Results:Baseline TCD4+ mRNA levels of IL-23 (P = 0.03) and TGF-β (P = 0.01) were different between healthy and infertile groups. However, IL-17, IL-4, IFN-γ, and IL-10 were expressed similarly regardless of fertility status. Comparing mRNA expression before and after SP exposure, our results have shown that relative expression of IL-23 significantly increased in successful (P = 0.04) and unsuccessful IVF groups (P = 0.01), whereas IL-10 expression increased only in the IVF failure group (P = 0.01).Conclusion:SP can make a positive effect on IVF outcome through alteration in CD4 + T-cell-related cytokines expression, especially IL-10 and IL-23.

Hippocampal astrocyte dysfunction contributes to etomidate-induced long-lasting synaptic inhibition

Cognitive impairments following the use of general anesthetics are well documented but the underlying mechanisms are unclear. Here, long-lasting cognitive deficits were observed in aged mice following administration of etomidate at a clinically relevant concentration (20 mg/kg); these deficits were closely related to hippocampal synaptic inhibition and astrocyte dysfunction. Using microdialysis and magnetic-activated cell-sorting techniques, we found that astrocyte secretion of glutamate, d-serine, and ATP, as well as astrocyte function, were depressed in the hippocampus following treatment with etomidate. Interestingly, hippocampal astrocyte inhibition (designer receptors exclusively activated by designer drugs; DREADDs) had no effect on the initial synaptic inhibition, but reversed synaptic and cognitive depression in the long term. Furthermore, continual activation of hippocampal astrocytes following administration of a sedative dose (8 mg/kg) of etomidate induced synaptic inhibition and cognitive dysfunction. Our results indicate that general anesthetic-induced hippocampal astrocyte dysfunction plays a role in maintaining synaptic inhibition, which eventually induces long-lasting cognitive deficits.

High glucose condition limited the angiogenic/cardiogenic capacity of murine cardiac progenitor cells in in vitro and in vivo milieu.

Murine c-kit+ cardiac cells were isolated and enriched by magnetic activated cell sorting technique. c-kit+ cells viability and colony-forming activity were evaluated by MTT and clonogenic assay. c-kit+ cells were exposed to endothelial, pericyte, and cardiomyocyte induction media containing 30mM glucose for 7days. We monitored the level of endothelial (VE-cadherin, CD31, and vWF), pericyte (NG2 , α-SMA, and PDGFR-β), and cardiomyocyte markers (cTnT) using flow cytometry, real-time Polymerase Chain Reaction (PCR), and Enzyme-Linked Immunosorbent Assay (ELISA) analyses. Ultrastructural changes were studied by transmission electron microscopy (TEM) in cells treated with 5-Azacytidine and 30mM glucose. Matrigel plug assay was performed to determine the angio/cardiogenic property of c-kit+ cells in a diabetic mouse model. Glucose of 30mM decreased c-kit+ cells viability and clonogenicity (P<0.05). The transdifferentiation capacity of c-kit+ cells into the endothelial lineage, pericytes, and cardiomyocytes were reduced through the inhibition of related genes (P<0.05). TEM analysis revealed cardiomyocyte differentiation rate in c-kit+ cells coincided with an increased intracellular lipid accumulation and reduced number of mitochondria. Similar to in vitro condition, the angiogenic capacity of c-kit+ cells was aborted in vivo indicated by reduced NG2 , α-SMA, CD31, and vWF levels. High glucose condition reduces the angio/cardiogenic capacity of cardiac c-kit+ cells in vitro and in vivo. SIGNIFICANCE OF THE STUDY: High glucose condition seen in diabetes mellitus could affect the regenerative potential of cardiac tissue. The current experiment showed that the exposure of murine cardiac progenitor cells (CD117+ cells) to condition containing 30mM glucose could decrease the differentiation properties into endothelial cells, pericytes, and mature cardiomyocytes in vitro and in vivo. Our finding confirmed that the angiogenic/cardiogenic potential cardiac progenitor cells decrease under treatment with high glucose content as seen in the diabetic condition.

A microfluidic microwell device for immunomagnetic single-cell trapping

Single-cell analysis has been widely applied in various biomedical applications, such as cancer diagnostics, immune status monitoring, and drug screening. To perform an accurate and rapid cellular analysis, various magnetic-activated cell sorting techniques are available in the markets. However, large sample requirement and uneven magnetic field distribution limit its application in single-cell trapping and following analysis. To address these problems, we developed a microfluidic microwell device for immunomagnetic single-cell trapping. By adding a microwell layer between the microchannel and magnet, the magnetic field along the device becomes more uniform. Besides, magnetic beads can be retained in the array of microwell after the high-speed washing step, whereas untrapped beads would be flushed away, resulting in high single-particle trapping efficiency (62%) and purity (99.6%). To achieve large-area single-cell trapping, we introduced a “sweeping” loading protocol to further expand the single-particle trapping range. In the microwell region near to the edge of the magnet, over 3000 single magnetic beads were trapped in a 10 mm2 area. Finally, we demonstrated immunomagnetic-labeled THP-1 cells can successfully be trapped at single-cell level in the microwell. The cell trapping process can be done in 10 min. We believe the platform with an accurate and efficient single-cell trapping functionality could potentially be used for various cellular analyses at the single-cell level.

Evaluation of EBV transformation of human memory B-cells isolated by FACS and MACS techniques

Several studies have been performed to develop effective neutralizing monoclonal antibodies. The Epstein–Barr virus (EBV) can efficiently immortalize B-cells to establish lymphoblastoid cell lines (LCL) and so it has been used extensively for transformation of B-cells to produce and secrete immunoglobulin. The present study addressed the effect of TLR7/8 agonist (R848), feeder cells layer and fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) cell separation methods on the transformation efficiency of antibody-producing memory B-cells. For these studies, the antigen used for analyses of antibody formation was the tetanus neurotoxin (TeNT) derived from Clostridium tetani. The results here showed that employing an HFFF.PI6 feeder cell layer, R848 agonist and FACS-mediated purification of memory B-cells led to increased transformation efficiency. Altogether, the effects of the R848 and the feeder cells provided an efficient method for EBV transformation of human B-cells. Moreover, there was an advantage in using FACS sorting of B-cells over the MACS method in the context of EBV transformation and immortalization of precursors of antigen-specific B-cells.

Analysis of human B cell response to recombinant Leishmania LPG3.

To evaluate the capability of recombinant Leishmania LPG3 and its fragments in the activation of B cells. In the present study, human B cells were purified from peripheral blood of 10 adult healthy subjects using magnetic-activated cell sorting technique. Subsequently, purified B cells were treated with recombinant LPG3, and its N-terminal and C-terminal fragments at different concentrations (2, 10 and 20μg/mL). B cell activation was assessed through expression of CD69 molecule by flow cytometry and secretion of IL-6, TNF-α and IL-10 cytokines via enzyme-linked immunosorbent assay following treatment with recombinant antigens. Our results showed that while the recombinant LPG-3 could significantly increase the production of IL-6 and TNF-α (P<0.05) in B cells, it had no effect on the secretion of IL-10 by B cells. Our study indicated that recombinant LPG-3 and especially its N-terminal fragment could stimulate B cell response as an important immune response component against leishmaniasis. Thus, it seems that it can be considered as an effective adjuvant in vaccine developments against leishmaniasis.

Recurrent Abnormal Clones in Myelodysplastic Syndrome Marrow Originate from Cells at a Pluripotent Stem Level and Maintain Their Early Differentiation Potency

Objective: The present study aimed to investigate the origins and differentiation potencies of 4 common malignant clonal cell types (+8, 5q−/−5, 20q−/−20, 7q−/−7) in myelodysplastic syndrome (MDS) and to investigate whether the trisomy of chromosome 8 occurs subsequently to other chromosomal abnormalities. Methods: The present study analyzed a total of 46 cases of chromosomal abnormalities in MDS patients. The magnetic activated cell sorting technique (MACS) was used to sort the CD34+CD38− (pluripotent hematopoietic stem cells) and CD34+CD38+ cells (committed progenitor cells) from the bone marrow mononuclear cells (BMNCs) of these patients; the sorted cells were then individually smeared. Meanwhile, cytospins were prepared from the remaining CD34− BMNCs after cell sorting. The clonal cell proportions in these three types of smears were detected by fluorescence in situ hybridization (FISH). Cases in which +8 was associated with another abnormality (2 cases each in combination with abnormalities in chromosomes 7, 5, and 20) were dually hybridized with the cep8 probe and another corresponding probe. Results: (1) for abnormalities of +8, 5q−/−5, 20q−/−20 or chromosome 7 involvements, clonal cells above the baseline level were detected in the pluripotent stem cell level. (2) The average clonal cell proportion in the committed progenitor cells of the 46 cases increased to 75.3% from 57.3% at the level of stem cell (p < 0.001). The groups with +8 and chromosome 5 abnormalities showed a statistically significant increase in clonal cells at the progenitor cell stage. At the individual level, 33 of 46 cases showed significant increases in clonal cells at the progenitor cell stage relative to the stem cell stage, whereas the clonal cell proportion in the CD34− BMNCs generally did not increase relative to the committed progenitor cell population. (3) The dual hybridization analysis showed that if +8 and another abnormality were present in the same abnormal clone according to G-banding, +8 always coexisted with the other chromosomal abnormality at the single cell level; there were no situations in which +8 occurred later than the other chromosomal abnormality. Conclusion: It seems that the all malignant MDS clones originated at the pluripotent hematopoietic stem cell stage and that the proliferation and differentiation potencies were retained partly in these clonal cells. The present study failed to confirm that the trisomy 8 occurred subsequently to the other abnormalities, but some in vitro or transplant experiments maybe prove the succession of clonal origination.

Oxidized low‐density lipoprotein induces hematopoietic stem cell senescence

We have investigated oxidized low-density lipoprotein (ox-LDL) induced senescence in hematopoietic stem cells (HCs). Mouse Sca-1+ HCs were separated and purified using the magnetic activated cell sorting technique. Ox-LDL induced significant senescence in HCs measured by SA-β-Gal staining, and reduced CFU-Mix colony-forming capacity, arresting cells at G0/G1 phase. In agreement with the cell cycle arrest, ox-LDL markedly reduced the expression of CDK4, cyclin D, and cyclin E. As possible contributing factors for cell senescence, ox-LDL also induced cellular oxidative stress and reduced telomerase activity.

BCL11B suppression does not influence CD34+ cell differentiation and proliferation

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B) gene plays a critical role in T-cell differentiation and proliferation. However, little is understood about the role of BCL11B in human hematopoietic stem/progenitor cells. Small interfering RNA (siRNA)-mediated suppression of the BCL11B was shown to induce apoptosis in human T-cell acute lymphoblastic leukemia cells. To further characterize the role of BCL11B in hematopoietic stem/progenitor cells and assess the safety of siRNA-mediated targeted therapy, the in vitro differentiation and proliferation of CD34+ cells after BCL11B-siRNA935 treatment were studied. CD34+ cells were sorted from three cases of umbilical cord blood by the magnetic activated cell sorting technique, and the purity was identified by flow cytometry. BCL11B-siRNA935 was delivered into CD34+ cells by nucleofection and the BCL11B expression level was analyzed by quantitative real-time polymerase chain reaction. Erythroid burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and megakaryocyte colony-forming units (CFU-Meg) were assessed using BCL11B-siRNA935-treated CD34+ cells by the methylcellulose semi-solid culture method. The BCL11B expression level in CD34+ cells was significantly lower than that in Molt-4 cells and peripheral blood mononuclear cells from healthy individuals. An approximate one-fold reduction in the BCL11B mRNA level was observed 24 hours post-transfection with BCL11B-siRNA935. However, there was no significant difference on the colony formation ability of BFU-E, CFU-GM, and CFU-Meg for CD34+ cells between the BCL11B-siRNA935-treated and mock-transfected groups (P > 0.05). BCL11B suppression by RNA interference had no significant influence on the differentiation and proliferation of CD34+ cells. In conclusion, the BCL11B-siRNA935 used in this study may be safe, and BCL11B may be considered a new candidate for targeted gene therapy in T-cell malignancies.

New Minimal Immunosuppression Strategies for Composite Tissue Allograft Transplantation: The Cleveland Clinic Experience

Clinical application of composite tissue allografts requires the introduction of new minimal immunosuppressive or tolerance-inducing protocols. We have established a protocol for tolerance induction under α/β selective blocking antibody T-cell receptor combined with interleukin-2 blocker cyclosporin A therapy. Application of this therapy resulted in induction of tolerance in fully allogenic and semiallogenic limb allograft transplants. Based on this experience we have found that the bone marrow component of the allograft is essential for tolerance induction and have confirmed that injection of bone marrow of donor origin directly into the bone marrow compartment of allograft recipients resulted in higher chimerism levels. In addition, we have developed a system for isolation of chimeric cells, which are specific for both the donor and recipient, by using magnetic activated cell sorting technique and flow cytometry. These new, experimental approaches confirm that introducing minimal suppression to patients undergoing hand or face transplantation will likely be achieved in the near future.

CpG methylation modulates tissue-specific expression of a transgene in chickens

The use of genetically modified germ cells is an ideal system to induce transgenesis in birds; the primordial germ cell (PGC) is the most promising candidate for this system. In the present study, we confirmed the practical application of this system using lentivirus-transduced chicken gonadal PGCs (gPGCs). Embryonic gonads were collected from 5.5-d old Korean Oge chickens (black feathers). The gPGC population was enriched (magnetic-activated cell sorting technique) and then they were transduced with a lentiviral vector expressing enhanced green fluorescent protein (eGFP), under the control of the Rous sarcoma virus (RSV) promoter. Subsequently, the eGFP-transduced PGCs were transplanted into blood vessels of 2.5-d-old embryonic White Leghorn (white feathers). Among 21 germline chimeric chickens, one male produced transgenic offspring (G 1 generation), as demonstrated by testcross and genetic analysis. A homozygous line was produced and maintained through the G 3 generation. Based on serum biochemistry, there were no significant physiological differences between G 3 homozygotes and non-transgenic chickens. However, since eGFP transgene expression in G 3 chickens varied among tissues, it was further characterized by Western blotting and ELISA. Furthermore, there were indications that DNA methylation may have affected tissue-specific expression of transgenes in chickens. In conclusion, the PGC-mediated approach used may be an efficient tool for avian transgenesis, and transgenic chickens could provide a useful model for investigating regulation of gene expression.

Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Although adult bone-marrow-derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non-dental stem-cell-mediated odontogenesis remain an open question. STRO-1(+) (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic-activated cell-sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2-day-old rat incisors. Co-cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up-regulated alkaline phosphatase activity, cell-cycle modification, and the expression of tooth-specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth-shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin-pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin-like structures and fibrous connective tissues respectively. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non-dental stem cell populations.

Activation pattern of caspases in human spermatozoa

Objective We investigated the activation of caspases 8, 9, 1, and 3 in human ejaculated spermatozoa to study main pathways of apoptosis. Potential functional impact of this phenomenon and possible activation mechanisms were examined by [1] subjecting cells to freezing and thawing, and [2] testing the dependence of caspase activity on membrane integrity. Design Experimental study. Setting Andrology Center Leipzig (European Academy of Andrology). Patient(s) Ten healthy volunteers, 40 semen samples. Intervention(s) Sperm populations were separated by annexin V magnetic activated cell sorting (MACS) according to externalized phosphatidylserine. Main outcome measure(s) Active caspases 8, 9, 1, and 3 were examined in human ejaculated spermatozoa before and after cryopreservation. Result(s) The active caspases were localized in the postacrosomal region (caspases 8, 1, and 3) or in the midpiece (caspase 9). Cryopreservation led to a significant increase in the number of cells showing activation of all caspases. The MACS separation resulted in a significant depletion of sperm positive for activated caspases within the externalized phosphatidylserine–negative fraction. Conclusion(s) Caspases 8, 9, 1, and 3, representing the main pathways of apoptosis, are present in human spermatozoa and can become activated, particularly after freezing and thawing, and are associated with changes in the outer cell membrane. Spermatozoa showing these changes and caspase activation can be efficiently removed from semen samples by the annexin V MACS technique.

Nurture vs nature: how can we optimize sperm quality?

Nurture vs nature: how can we optimize sperm quality?