Lymphoid Lineage γδ T Cells Were Successfully Generated from Human Pluripotent Stem Cells via Hemogenic Endothelium.

γδ T cells are a rare and unique prototype of T cells that share properties with natural killer cells in secondary lymphoid organs. Although many studies have revealed the function and importance of adult-derived γδ T cells in cancer biology and regenerative medicine, the low numbers of these cells hamper their application as therapeutic cell sources in the clinic. To solve this problem, pluripotent stem cell-derived γδ T cells are considered alternative cell sources; however, few studies have reported the generation of human pluripotent stem cell-derived γδ T cells. In the present study, we investigated whether lymphoid lineage γδ T cells were successfully generated from human pluripotent stem cells via hemogenic endothelium under defined culture conditions. Our results revealed that pluripotent stem cells successfully generated γδ T cells with an overall increase in transcriptional activity of lymphoid lineage genes and cytolytic factors, indicating the importance of the optimization of culture conditions in generating lymphoid lineage γδ T cells. We uncovered an initial step in differentiating γδ T cells that could be applied to basic and translational investigations in the field of cancer biology. Based on our result, we will develop an appropriate method to purify γδ T cells with functionality and it helpful for the study of basic mechanism of γδ T cells in pathophysiologic condition as well as clinic application.

Clinical Assessment of Intravenous Endothelial Progenitor Cell Transplantation in Dogs.

Endothelial progenitor cells (EPCs) have been applied for cell therapy because of their roles in angiogenesis and neovascularization in ischemic tissue. However, adverse responses caused by EPC therapy have not been fully investigated. In this study, a human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated using Ficoll-Hypaque. There were four experimental groups: 10 ml saline infusion group (injection rate; 3 ml/min), 10 ml saline bolus group (injection rate; 60 ml/min), 10 ml EPCs infusion group (2 x 105 cells/ml, injection rate; 3 ml/min), 10 ml EPCs bolus group (2 × 105 cells/ml, injection rate; 60 ml/min). Clinical assessment included physical examination and laboratory examination for intravenous human EPC transplantation in dogs. The results revealed no remarkable findings in vital signs among the dogs used. In blood analysis, platelet counts in saline infusion groups were significantly higher than in the EPC groups within normal ranges, and no significant differences were observed except K+, Cl- and blood urea nitrogen/urea. In ELISA assay, no significant difference was observed in serum tumor necrosis factor alpha. The serum concentration of vascular endothelial growth factor was significantly higher in EPC groups than in saline groups, and interleukin 10 was significantly up-regulated in the EPC infusion group compared with other groups. In conclusion, we demonstrated that no clinical abnormalities were detected after intravenous transplantation of human EPCs in dogs. The transplanted xenogenic EPCs might be involved in anti-inflammatory and angiogenic functions in dogs.

Comparison of complex regional pain syndrome and fibromyalgia: Differences in beta and gamma bands on quantitative electroencephalography.

Complex regional pain syndrome (CRPS) and fibromyalgia (FM) share many features. Both can cause severe pain and are considered to have a mechanism of action, including dysfunction of the sympathetic nervous system. However, they have clinical differences in pain range and degree. The present study aimed to find neurophysiologic differences between CRPS and FM using quantitative electroencephalography (QEEG). Thirty-eight patients with CRPS and 33 patients with FM were included in the analysis. Resting-state QEEG data were grouped into frontal, central, and posterior regions to analyze for regional differences. General linear models were utilized to test for group differences in absolute and relative powers. As a result, the CRPS group relative to FM group showed lower total absolute powers in the beta band (F = 5.159, P < .05), high beta (F = 14.120, P < .05), and gamma band (F = 15.034, P < .05). There were no significant differences between 2 groups in the delta, theta, and alpha bands. The present findings show that the CRPS and FM groups differ mainly in the high frequency, which may reflect their distinct pathophysiology and symptomatology. Our study suggests that the QEEG differences can be clinically useful in assessing brain function in patients with CRPS and FM.

Increased Frontal Gamma and Posterior Delta Powers as Potential Neurophysiological Correlates Differentiating Posttraumatic Stress Disorder from Anxiety Disorders.

OBJECTIVE: Posttraumatic stress disorder (PTSD) is distinct from anxiety disorders in its etiology and clinical symptomatology, and was reclassified into trauma- and stressor-related disorders in DSM-5. This study aimed to find neurophysiological correlates differentiating PTSD from anxiety disorders using resting-state quantitative electroencephalography (qEEG). METHODS: Thirty-six patients with either PTSD or acute stress disorder and 79 patients with anxiety disorder were included in the analysis. qEEG data of absolute and relative powers and patients' medication status on the day of qEEG examination were obtained. Electrodes were grouped into frontal, central, and posterior regions to analyze for regional differences. General linear models were utilized to test for group differences in absolute and relative powers while controlling for medications. RESULTS: PTSD patients differed from those with anxiety disorders in overall absolute powers [F(5,327)=2.601, p=0.025]. Specifically, overall absolute delta powers [F(1,331)=4.363, p=0.037], and overall relative gamma powers [F(1,331)=3.965, p=0.047] were increased in PTSD group compared to anxiety disorder group. Post hoc analysis regarding brain regions showed that the increase in absolute delta powers were localized to the posterior region [F(1,107)=4.001, p=0.048]. Additionally, frontal absolute gamma powers [F(1,107)=4.138, p=0.044] were increased in PTSD group compared to anxiety disorder group. CONCLUSION: Our study suggests increased overall absolute delta powers and relative gamma powers as potential markers that could differentiate PTSD from anxiety disorders. Moreover, increased frontal absolute gamma and posterior delta powers might pose as novel markers of PTSD, which may reflect its distinct symptomatology.

Despite the donor's age, human adipose-derived stem cells enhance the maturation and development rates of porcine oocytes in a co-culture system.

The paracrine interactions between cumulus-oocyte complexes (COCs) and follicular somatic cells during in vitro maturation (IVM) were investigated. To optimize IVM conditions, many studies have applied exogenous growth factors and cell feeding/co-culture systems using various cell types to replicate the natural follicular microenvironment during IVM. A potential candidate as cell feeders is adipose-derived stem cells (ASCs) which secrete high levels of growth factors that have roles in oocyte maturation. However, the cell donor's age should be considered because biological aging also occurs in stem cells. In the present study, the contributions of ASCs from young and old donors on an IVM co-culture system were analyzed by comparing the oocyte maturation rate, cumulus expansion index, preimplantation development after parthenogenetic activation (PA), and expression of growth factor signaling genes related to oocyte maturation in ASCs, oocytes and cumulus cells under the same culture conditions. Our study demonstrated that the confluence, viability and cell size of ASCs between young and old donors were not significantly different and only the Fibroblast Growth Factor 2 (FGF2) signaling gene showed higher expression in ASCs from young donors. The oocyte maturation rate in the young donor group (87.8 ±â€¯1.2%) was significantly higher than in the old donor (81.1 ±â€¯2.1%) and control (73.8 ±â€¯2.1%) groups. After IVM, most gene expression levels in oocytes and cumulus cells in the co-culture groups were higher than in the control but the apoptotic ratios were reduced. The blastocyst development rates were not different between the young and old donor groups (23.9 ±â€¯1.3% and 20.7 ±â€¯0.8%, respectively) but the percentages were higher in both groups compared to the control group (16.4 ±â€¯1.2%). A similar pattern was also found for blastocyst total cell numbers in that the young donor group (87.5 ±â€¯5.2 cells) was not different than the old donor group (77.5 ±â€¯3.4 cells) but both groups exhibited higher number of cells compared with the control group (57.9 ±â€¯6.0 cells, p < .05). Our study strongly suggested that the co-culture IVM system with ASCs greatly improved the maturation and development rates of porcine oocytes. Moreover, ASCs from young donors more effectively supported porcine oocyte maturation than those from old donors although this difference did not translate into improved developmental competence.

Suberoylanilide hydroxamic acid during in vitro culture improves development of dog-pig interspecies cloned embryos but not dog cloned embryos.

This study was conducted to investigate whether the treatment of dog to pig interspecies somatic cell nuclear transfer (iSCNT) embryos with a histone deacetylase inhibitor, to improve nuclear reprogramming, can be applied to dog SCNT embryos. The dog to pig iSCNT embryos were cultured in fresh porcine zygote medium-5 (PZM-5) with 0, 1, or 10 µM suberoylanilide hydroxamic acid (SAHA) for 6 h, then transferred to PZM-5 without SAHA. Although there were no significant differences in cleavage rates, the rates of 5-8-cell stage embryo development were significantly higher in the 10 µM group (19.5 ± 0.8%) compared to the 0 µM groups (13.4 ± 0.8%). Acetylation of H3K9 was also significantly higher in embryos beyond the 4-cell stage in the 10 µM group compared to the 0 or 1 µM groups. Treatment with 10 µM SAHA for 6 h was chosen for application to dog SCNT. Dog cloned embryos with 0 or 10 µM SAHA were transferred to recipients. However, there were no significant differences in pregnancy and delivery rates between the two groups. Therefore, it can be concluded that although porcine oocytes support nuclear reprogramming of dog fibroblasts, treatment with a histone deacetylase inhibitor that supports nuclear reprogramming in dog to pig iSCNT embryos was not sufficient for reprogramming in dog SCNT embryos.

Effect of co-culture human endothelial progenitor cells with porcine oocytes during maturation and subsequent embryo development of parthenotes in vitro.

Human endothelial progenitor cells (EPCs) have been applied to regenerative medicine for their roles in angiogenesis as well as neovascularization, and these angiogenetic functions have beneficial effects on maturation of ovarian follicles. However, little information is available on whether EPCs on culture systems affect oocyte maturation and subsequent embryo development. Therefore, the objective of this study was to investigate the effect of EPC co-culture on porcine oocytes during in vitro maturation (IVM) and subsequent embryo development, and to examine gene expression in cumulus cells, oocytes and blastocysts. The effect of co-culture using EPC on porcine oocyte IVM was investigated. Oocytes were activated using electrical stimulation and embryo developmental competence was estimated. The expression of the genes related to cumulus expansion, oocyte maturation, embryo development, and apoptosis were analyzed. In result, there was a significantly increased maturation rate in EPC group compared with control (p < 0.05). Also, oocytes co-cultured with EPCs exhibited significantly improved blastocyst formation rates (p < 0.05). The expression of mRNAs associated with cumulus expansion and apoptosis in cumulus cells was significantly up-regulated in EPC group. Also, markedly increased levels of GDF9, BMP15, and BCL2 were observed in oocytes from the EPC group. Blastocysts in the co-culture group showed significantly higher SOX2, OCT4, and NANOG levels. In conclusion, co-culturing porcine oocytes with EPCs improves their maturation by regulating genes involved in cumulus cell expansion, oocyte maturation, and apoptosis. Moreover, EPC co-culture during IVM enhanced embryo development as shown by increased blastocyst formation rate and pluripotency-related gene expression.

Clinical assessment after human adipose stem cell transplantation into dogs.

Adipose tissue-derived stem cell (ASCs) are an attractive source of stem cells with therapeutic applicability in various fields for regenerating damaged tissues because of their stemness characteristics. However, little has reported on evaluating adverse responses caused by human ASC therapy. Therefore, in the present study, a clinical assessment after human ASC transplantation into dogs was undertaken. A total of 12 healthy male dogs were selected and divided into four groups: saline infusion, saline bolus, ASC infusion, and ASC bolus groups. Physical assessment and blood analysis were performed following ASC transplantation, and the concentrations of angiogenic factors, and pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). There were no adverse vital sign responses among the dogs. Blood analyses revealed no remarkable complete blood count or serum chemistry results. ELISA results for angiogenic and anti-inflammatory factors including matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were significantly higher in the two ASCs groups than in the controls. In conclusion, this study demonstrated that transplantation of human ASCs produced no adverse effects and could be used safely in dogs. In addition, human ASCs could be involved in modulating secretions of angiogenic factors including MMP9, VEGF, bFGF, and HGF and anti-inflammatory factor IL-10.

Effect of co-culture canine cumulus and oviduct cells with porcine oocytes during maturation and subsequent embryo development of parthenotes in vitro.

In the estrus stage, canine oocytes are surrounded by cumulus cells and undergo maturation in the oviduct for 2-3 days after ovulation. We hypothesized that canine oviduct cells (cOC) and canine cumulus cells (cCC) during this stage might affect the maturation of oocytes and thereby improve subsequent embryo development. Therefore, the objective of this study was to compare the effects of a cOC and cCC co-culture on oocyte in vitro maturation (IVM) and subsequent embryo development, and to analyze the gene expressions in a molecular fashion what co-culture actually gives the specific pathways in which the co-culture cells act to improve maturation and embryo development. The effect of co-culture using cOC and cCC on porcine oocyte IVM was investigated. Thereafter, oocytes were activated using electrical stimulation and embryo developmental competence was estimated. The expression of the genes related to oocyte maturation, embryo development and apoptosis were analyzed. Also, reactive oxygen species (ROS) levels after IVM was analyzed. The IVM rate and embryo development including cleavage, blastocyst formation rates, and total blastocyst cell numbers from cOC group were significantly higher than other groups (P < 0.05). The expression of SMAD2/3 and growth differentiation factor 9 (GDF9) was significantly increased in cOC and oocytes from the cOC group compared with other groups. Moreover, the levels of GDF9, prostaglandin-endoperoxide synthase 2 (PTGS2), WNT3A and matrix metalloproteinase 2 (MMP2) were significantly up-regulated in blastocysts from the cOC group. The concentration of ROS was significantly lower in the supernatant of cOC groups compared with other groups. Also, the expression of BCL2 was significantly increased in porcine cumulus cells and oocytes from cOC group. The present study demonstrated that co-culture with cOC improved in vitro porcine oocyte maturation and subsequent embryo development competence. Also, co-culture with cOC during IVM induces a suitable environment for oocyte maturation by enhancing the mRNA level of SMAD2/3 and GDF9, and for embryo development by elevating the expression level of PTGS2, WNT3A and MMP2. In addition, the decreased ROS level in cOC co-culture could have a beneficial influence on oocyte maturation.

Birth of clones of the world's first cloned dog.

Animal cloning has gained popularity as a method to produce genetically identical animals or superior animals for research or industrial uses. However, the long-standing question of whether a cloned animal undergoes an accelerated aging process is yet to be answered. As a step towards answering this question, we compared longevity and health of Snuppy, the world's first cloned dog, and its somatic cell donor, Tai, a male Afghan hound. Briefly, both Snuppy and Tai were generally healthy until both developed cancer to which they succumbed at the ages of 10 and 12 years, respectively. The longevity of both the donor and the cloned dog was close to the median lifespan of Afghan hounds which is reported to be 11.9 years. Here, we report creation of 4 clones using adipose-derived mesenchymal stem cells from Snuppy as donor cells. Clinical and molecular follow-up of these reclones over their lives will provide us with a unique opportunity to study the health and longevity of cloned animals compared with their cell donors.

Oocyte maturation-related gene expression in the canine oviduct, cumulus cells, and oocytes and effect of co-culture with oviduct cells on in vitro maturation of oocytes.

PURPOSE: In contrast to most other mammals, canine oocytes are ovulated in an immature state and undergo oocyte maturation within the oviduct during the estrus stage. The aim of the study was to investigate whether oviduct cells from the estrus stage affect the maturation of oocytes and show gene expression patterns related to oocyte maturation. METHODS: We analyzed MAPK1/3, SMAD2/3, and BMP6/15 expression in oviduct cells, cumulus cells, and oocytes from anestrus, estrus, and diestrus stages. Next, we investigated the effect of co-culture with oviduct cells derived from the estrus stage upon in vitro maturation (IVM) of canine oocytes. RESULTS: There was significantly higher MAPK1/3 (1.42 ± 0.02 and 2.23 ± 0.06), SMAD2/3 (0.77 ± 0.03 and 2.39 ± 0.07), and BMP15 (2.21 ± 0.16) expression in oviduct cells at the estrus stage (P < 0.05). In cumulus cells, MAPK1 (1.26 ± 0.07), SMAD2/3 (0.82 ± 0.01, 1.04 ± 0.01), and BMP6 (13.09 ± 0.11) expression was significantly higher in the estrus stage (P < 0.05). In oocytes, significant upregulation of MAPK1/3 (14,960 ± 3121 and 1668 ± 253.4), SMAD3 (774.6 ± 79.62), and BMP6 (8500 ± 895.4) expression was found in the estrus stage (P < 0.05). After 72 h of IVM culture, a significantly higher maturation rate was observed in oocytes co-cultured with oviduct cells (10.0 ± 1.5%) than in the control group (3.2 ± 1.4%). CONCLUSIONS: We demonstrate that oviduct cells at the estrus stage highly expressed MAPK1/3, SMAD2/3, and BMP15. Furthermore, canine oviduct cells from the estrus stage enhance the culture environment for canine oocyte maturation.

Spermine reduces reactive oxygen species levels and decreases cryocapacitation in canine sperm cryopreservation.

The objective of this study was to determine the ability of spermine to act as an antioxidant in scavenging reactive oxygen species (ROS), maintaining sperm function and decreasing cryocapacitation after cryopreservation. Although motility did not increase with spermine treatment, values for membrane integrity were significantly increased (P < 0.05). Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) indicated that spermine inhibits hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment group (P < 0.05). Higher expression of an anti-apoptotic gene (Bcl-2) and lower expression of a pro-apoptotic gene (Bax), together with decreased expression of the mitochondrial ROS modulator ROMO1, DNA repair due to oxidative damage (OGG1), spermine synthase (SMS), NADPH oxidase associated with motility (NOX5) and spermine amino oxidase (SMOX), all showed that 5.0 mM spermine treatment was beneficial to spermatozoa. Furthermore, the proportion of live spermatozoa with intact acrosomes after thawing in the treatment group was higher than in the control. After incubation in canine capacitating medium, numbers of live capacitated spermatozoa with reacted acrosomes were higher than in the control. Our results indicate that 5.0 mM spermine is an optimal concentration for maintaining sperm function, reducing ROS production, preventing apoptosis and adverse effects of cryocapacitation during canine sperm cryopreservation.

Cloning of the short-tailed Gyeongju Donggyeong dog via SCNT: conserving phenotypic inheritance.

Somatic cell nuclear transfer is a useful tool to maintain genetic information of animals. The Gyeongju Donggyeong dog is a breed registered as natural monument in Korea. The unique feature of the Donggyeong dog is its tail, as the Donggyeong dog can be classified as either short tailed or tailless. The aim of this study was to preserve the Donggyeong dog's unique feature by cloning. Fibroblasts were obtained from a short-tailed Donggyeong dog. In vivo matured oocytes were enucleated, microinjected with a donor cell and fused electrically. Reconstructed embryos were transferred to six recipient dogs. One surrogate became pregnant, and one short-tailed Donggyeong dog was delivered. This study demonstrated that the phenotype of the Donggyeong dog could be conserved by somatic cell nuclear transfer.

Propagation of elite rescue dogs by somatic cell nuclear transfer.

The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6-dimethylaminopyridine (DMAP) (SOF-DMAP) or porcine zygote medium (PZM-5) with 1.9 mmol/L DMAP (PZM-DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF-DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6-DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.

Altering histone acetylation status in donor cells with suberoylanilide hydroxamic acid does not affect dog cloning efficiency.

Although dog cloning technology has been applied to conservation of endangered canids, propagation of elite dogs, and production of transgenic dogs, the efficiency of cloning is still very low. To help overcome this problem, we evaluated the effect of treating donor cells with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on dog cloning efficiency. Relative messenger RNA expressions of the bax1/bcl2 ratio and Dnmt1 in fibroblasts treated with different concentrations (0, 1, 10, 50 µM) of SAHA and durations (0, 20, 44 hours) were compared. Treatment with 1 µM for 20 hours showed significantly lower bax1/bcl2 and Dnmt1 transcript abundance. Acetylation of H3K9 was significantly increased after SAHA treatment, but H4K5, H4K8 and H4K16 were not changed. After SCNT using control or donor cells treated with SAHA, a total of 76 and 64 cloned embryos were transferred to seven and five recipients, respectively. Three fetuses were diagnosed in both control and SAHA-treated groups by ultrasonography 29 days after the embryo transfer, but there was no significant difference in the pregnancy rate (4.2% vs. 4.3%). In conclusion, although SAHA treatment as used in this study significantly decreased bax1/bcl2 and Dnmt1 transcripts of donor nuclei, as well as increased H3 acetylation, it was not enough to increase in vivo developmental competence of cloned dog embryos.

Maintaining canine sperm function and osmolyte content with multistep freezing protocol and different cryoprotective agents.

Cryopreservation procedures cause osmotic stress to spermatozoa following cryoinjury and reduce their content of osmolytes. Conventional method for cryoprotectant loading and dilution on canine semen freezing which could be categorized in single step protocol, makes decreasing in sperm performance such as motility, morphology and viability. Therefore, the objective of the present study was to determine if a multistep protocol using glycerol or ethylene glycol can be used to overcome the osmotic sensitivity of canine spermatozoa, and to identify osmolytes that were involved in regulation of osmotic stress. A multistep protocol, comprising serial loading and dilution of cryoprotective agents by dividing the total volume of extender into 4 steps (14%, 19%, 27%, and 40%) every 30s, was compared to a single step method. Frozen-thawed spermatozoa in the multistep group showed superior quality (P<0.05) compared with the single step process in progressive motility (23.3 ± 1.3% vs. 12.5 ± 1.6%), intact membranes (66.5 ± 2.8% vs. 49.5 ± 2.6%) and bent tail (29.2 ± 3.2% vs. 46.2 ± 1.9%). Multistep also succeeded in minimizing loss of the osmolytes carnitine (20.6 ± 2.0 nmol/U G6PDH vs. 10.8 ± 2.1 nmol/U G6PDH) and glutamate (18.4 ± 1.6 nmol/U G6PDH vs. 14.4 ± 0.8 nmol/U G6PDH) compared to the single step group. Moreover, glycerol with multistep was more advantageous for maintaining sperm quality than ethylene glycol. In conclusion, the multistep protocol with glycerol can be used to improve the morphology, motility and osmolytes content of frozen-thawed canine spermatozoa.

Improvement of cloned embryos development by co-culturing with parthenotes: a possible role of exosomes/microvesicles for embryos paracrine communication.

It is well known that embryos cultured in a group can create a microenvironment through secretion of autocrine and paracrine factors that can support and improve the embryos' development when compared to the embryos cultured individually. In this study, we used a co-culture system for paracrine communication between different kinds of embryos. The results showed that co-culture of porcine parthenogenetic (PA) embryos significantly improved the in vitro development of cloned (nuclear transfer, NT) embryos. To reveal the possible mechanism of communication between the two groups, we isolated exosomes/microvesicles (EXs/MVs) from the PA embryos conditioned medium (PA-CM) through differential centrifugation and identified them through transmission electron microscope and immunoflourescence against exosomal/membrane marker CD9. Furthermore, these EXs/MVs were found to contain mRNA of pluripotency genes (Oct4, Sox2, Klf4, c-Myc, and Nanog), and the PKH67-labeled EXs/MVs could be internalized by the NT embryos. The current study demonstrates that cloned embryos' developmental competence can be improved through co-culturing with PA embryos and revealed, for the first time, that in vitro-produced embryos can secrete EXs/MVs as a possible communication tool within their microenvironment. Moreover, it provides a new paradigm for embryo-to-embryo communication in vitro.