Time-lapse Imaging Technology Research Articles

Morphokinetics

Morphokinetics in IVF refers to the study of embryo development and quality based on the observation of their changing morphology over time. It involves closely monitoring the embryo’s cell division patterns, size, and other features using time-lapse imaging technology. While morphokinetics has shown promise in improving embryo selection based on morphological grading and provides novel kinetic parameters, it also remains surrounded by controversy. The debate on the use of time-lapse includes factors such as ethical concerns, cost and time, over selection of embryos, misinterpretation of data, and limited evidence. It is argued that there is a lack of strong scientific evidence demonstrating the superiority of morphokinetics over traditional methods of embryo development which includes grading of the embryos using a standardized grading system. The debate has been prolonged by the fact that studies show conflicting results, leading to questions about its reliability. Moreover, there are also ethical concerns about the time-lapse technology. There has been a long-standing discussion in ART regarding the use of add-ons in a treatment cycle. Time-lapse is often offered as an add-on in some clinics. This raises questions about accessibility, financial burden, risk of giving false hope to the patients, as well as distraction from the core treatment. In conclusion, the debate regarding the use of morphokinetics in the form of time-lapse imaging remains unsettled mainly due to conflicting data. The controversy will only be resolved as more information will come to light and studies will show concrete evidence of improved ART results credited to the use of morphokinetics.

Research progress of time-lapse imaging technology and embryonic development potential: A review.

Cultivation and selection of high-quality human embryos are critical for the success of in vitro fertilization-embryo transfer. Time-lapse imaging technology (TLI) provides a stable culture environment for embryos, which can continuously observe and record the development process of early embryos, so that doctors can record embryo development time parameters more accurately. In this study, we review the current observation and research on the main embryo dynamics parameters in TLI and discusses their significance and development for embryo development potential. To analysis and summary, the application and research situation of TLI, we searched PubMed, Web of Science, and China National Knowledge Infrastructure, using TLI, embryo dynamics parameters, embryo development potential as Keywords, cited 50 out of the initial 89 selected literatures and summarized. With comparative analysis and research, we found that the embryo dynamic parameters provided by TLI has been intensively studied in clinical empirical and observational research, extensive experimental data verified its effectiveness and advantages in embryo development potential assessment. TLI provides technical support of embryo dynamic parameters, which may become the quantitative indicators for superior embryos and pregnancy prediction as well. Existing studies have shown that certain kinetic parameters provided by TLI culture can predict embryo implantation, but no parameter has been confirmed as the absolute correlation biological indicators yet. In this review we believe that further research is needed to verify these preliminary and sometimes contradictory results, and explore the predictive significance of various embryo kinetic parameters relying on TLI technology for embryo development potential.

Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence in vitro: a preliminary assessment using time-lapse imaging.

The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm2, respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.

P–132 Centralized versus local embryologists scoring of blastocyst quality obtained in a large European multicenter clinical trial

Abstract Study question Does blastocyst quality scoring by central assessment deviate from local assessment and potentially lead to the selection of a different single blastocyst for transfer? Summary answer Central and local assessment provided the same quality classification (poor / good / top) in 69% of all blastocysts and 63% of all transferred blastocysts. What is known already Blastocyst quality is scored most frequently by three morphological parameters, namely expansion and hatching (EH) status, inner cell mass (ICM) grading and trophectoderm (TE) grading. The score is used to define the quality classification (poor / good / top) which determines which embryo is to be transferred or cryopreserved. Blastocyst scoring and grading can be highly subjective, which does influence the choice for transfer and cryopreservation. Time-lapse imaging technology captures additional input about embryo development as well as enables centralized data storage and sharing for independent central assessments. Study design, size, duration Pooled embryo analysis from a prospective, randomized, multicenter trial (RAINBOW) of 619 women undergoing ovarian stimulation with an individualized dose of follitropin delta in a long GnRH agonist protocol between May 2018 and January 2020. Blastocysts were centrally assessed using time-lapse images by two independent assessors and one adjudicator . Selection of the blastocyst for transfer by local assessment was based on morphological scoring and not on morphokinetic time-lapse parameters. Participants/materials, setting, methods Oocytes were fertilized by ICSI and cultured in the Embryoscopeâ (Vitrolife) up to day 5 for transfer or day 5/6 for cryopreservation. Embryos were assessed as either non-blastocyst or blastocyst. Blastocysts were graded centrally and locally at 116 hrs of development, based on EH status (1–6), ICM (A-D) and TE grading (A-D). Central assessors were blinded to local assessment and embryo transfer selection. Main results and the role of chance In total 4282 embryos were assessed centrally, of which 2046 day 5 embryos (48%) were adjudicated due to a scoring difference of at least one parameter between the two central assessors. In total 38% of day 5 embryos were judged as non-blastocysts and 62% as blastocysts of which 61% (i.e. 38% of all embryos) were determined to be of good or top quality. Identical results in terms of quality classification (poor / good / top) were obtained for 69% of blastocysts between local and central assessment and in 78%, between the two central assessors. Moreover, central and local scoring were identical in 62% for EH status, 53% for ICM grading and 57% for TE grading. For all transferred blastocysts (n = 508), central and local quality assessment was aligned for 63%. The ongoing pregnancy rate following single blastocyst transfer (SBT) was 41% (202/489), and similar to when considering only the transfers for which the central assessment had the same or a higher classification than the local assessment (166/411=40%). In 16% of all SBT, central quality assessment gave a lower score for the transferred blastocyst than the central assessment. This discrepancy could potentially have led to transfer of a different blastocyst. Limitations, reasons for caution This trial included assessments made by embryologists from 20 IVF centres. Some centres has limited experience with time-lapse technology for morphological blastocyst scoring. Scoring could therefore have been affected by differences in focal planes, magnification and contrast compared to inverted microscopy, with potential influence on blastocyst scores and quality classification. Wider implications of the findings: Local and central blastocyst quality classification based on morphology aligns well but remains subjective. Embryo assessment may benefit from using tools like artificial intelligence-based algorithms for a more objective analysis. Trial registration number NCT03564509

O-208 High sperm DNA fragmentation index negatively impacts embryo morphokinetics, but not embryo morphology and development rates: the importance of time-lapse imaging system

Abstract Study question Can time-lapse imaging (TLI) identify morphokinetic events impacted by high sperm DNA fragmentation index (DFI), irrespective of conventional morphological embryo assessment and development rate? Summary answer Embryo morphokinetic parameters are negatively impacted by high DFI, whereas conventional morphological embryo assessment and blastocyst development rate are not related to DNA integrity. What is known already Paternal genome activation occurs late in the embryo, and therefore, the negative impact of sperm factors on embryo development is more often observed in the outcomes of pregnancy, such as embryonic implantation and pregnancy loss, than in the potential for embryonic development itself, such as successful development to the blastocyst stage. With the development of TLI technology, and the possibility of assessing complete embryonic development, we hypothesized that sperm factors related to DNA fragmentation may interfere with the speed and pattern of cell divisions, leading to slower embryos; something that would not be detected by conventional morphological embryo assessment. Study design, size, duration The study included 978 zygotes cultured until day five in a TLS incubator between March/2019 and August/2020, derived from 118 patients undergoing ICSI in a private university-affiliated IVF center. Kinetic markers from the point of insemination were recorded. Generalized linear models adjusted for potential confounders, followed by Bonferroni post hoc were used to compare timing of specific events in patients with low (&amp;lt;30%) of high (≥30%) DFI. The post hoc achieved power was &amp;gt; 90%. Participants/materials, setting, methods Recorded kinetic markers were: timing to pronuclei appearance and fading (tPNa and tPNf), timing to two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), and timing to morulae (tM), start of blastulation (tSB) and blastulation (tB). Durations of second and third cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1, s2, and s3 were calculated. The KIDScore ranking was also recorded. Main results and the role of chance Blastocyst development (53.1% ± 1.3 vs. 55.1% ± 1.5, p = 0.380) and high-quality rates (87.9% ± 2.9 vs. 86.2% ± 3.6, p = 0.749) were similar between embryos derived from sperm samples with &amp;lt;30% DFI (n = 592) and ≥30% DFI (n = 386), respectively. Embryos derived from sperm samples with ≥30% DFI showed significantly slower divisions compared to those from &amp;lt;30% DFI: tPNa (6.1h ± 0.2 vs. 6.8h ± 0.2, p = 0.030), tPNf (23.0h ± 0.3 vs. 24.2h ± 0.3, p = 0.009), t2 (25.4h ± 0.3 vs. 26.9h ± 0.3, p = 0.002), t3 (34.8h ± 0.3 vs. 37.3h ± 0.4, p &amp;gt; 0.001), t4 (37.5h ± 0.4 vs. 39.3h ± 0.4, p = 0.003), t5 (46.2h ± 0.5 vs. 49.5h ± 0.6, p &amp;lt; 0.001), t6 (49.7h ± 0.5 vs. 52.8h ± 0.6, p = 0.001), t7 (52.4h ± 0.6 vs. 55.6h ± 0.7, p = 0.001), t8 (56.2h ± 0.7 vs. 58.9h ± 0.8, p = 0.017), tSB (97.5h ± 1.5 vs. 105.9h ± 1.7, p = 0.002), tB (108.6h ± 0.8 vs. 112.4h ± 1.2, p = 0.016). The KIDScore ranked significantly different between embryos derived from samples with &amp;lt;30% or ≥ 30% DFI (4.5 ± 0.1 vs. 3.9 ± 0.2, p = 0.033, respectively). A significant difference was observed in implantation rate (&amp;lt;30% DFI: 51.5% ± 2.2 vs. ≥30% DFI: 30.5% ± 1.3, p &amp;lt; 0.001). Limitations, reasons for caution Retrospective nature of this study and the small sample size may be a reason for caution, despite adequate power has been achieved. Wider implications of the findings Increasing DFI correlates with delayed cell cleavage and blastulation, leading to reduced implantation rates, without compromising blastulation rate and quality. This finding highlights the importance of TLI for the identification and de-selection of slow-growing embryos for transfer, in cycles with high DFI. Trial registration number Not applicable

THE HISTORY, OPPORTUNITIES AND PROSPECTS OF TIME-LAPSE TECHNOLOGIES IN THE STUDY OF EARLY HUMAN EMBRYONIC DEVELOPMENT

Despite advances in assisted reproductive technologies, the high failure rate of existing stimulation protocols remains a key industry challenge. One of the leading reasons for this is the limited ability to assess the biological potential of the embryo and its chances of implantation. Over the past ten years, the focus of attention in reproductive technologies has significantly shifted from the patient to the embryo, since the need to improve their effectiveness stimulates the need to understand the deep processes of early development of the embryo. In order to increase the effectiveness of in vitro fertilization procedures in clinical embryology, high-tech methods of culturing and evaluating embryos are being introduced and improved. The purpose of the review is to demonstrate the history, possibilities and prospects in the study of early human embryonic development of time-lapse imaging technology. The active study and use of the capabilities of the time-lapse slow-motion technology allowed not only to expand the understanding of the processes of early development of the embryo, but also at the current moment allows us to assess its potential from the point of view of both biological and clinical perspectives. The main advantages of this method are the possibility of morphological assessment during the continuous cultivation of embryos in closed-type incubators without their extraction, as well as the determination of the exact time intervals of key events of the stages of embryo development with special attention to those moments that are not available for observation and fixation under conditions of traditional cultivation. clinical practice. The main point of growth for the development of time-lapse imaging technology was the creation and validation of the so-called morphokinetic criteria and algorithms for assessing the quality of developing embryos. The key perspective of the method is its use in combination with elements of artificial intelligence in order to predict the most potential embryo for transfer into the uterine cavity. Modern directions of research using the method of time-lapse shooting are the continuation of the development of morphokinetic algorithms and their effective criteria, the introduction of the technology of self-learning computer programs and the adaptation of these tools in clinical practice, the search and assessment of possible factors influencing the morphokinetics of embryos, quality control of the work of embryological laboratories. The future development of such technologies is presented in combination not only with the capabilities of artificial intelligence, but also in combination with the use of non-invasive genetic screening, the assessment of metabolomics and proteomics of developing embryos.

46 Use of time-lapse imaging technology to assess relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos with their developmental competence invitro

The assessment of morphology and digital image opacity may provide valuable information on embryo viability because such traits are linked to embryonic gene expression, metabolism and ultrastructure. Time-lapse imaging has been used in research to monitor the dynamic nature of the developing pre-implantation embryo, which includes capturing alterations in various morphological parameters over time. The present study examined the effectiveness of time-lapse technology in assessing several morphometric and phototextural parameters for predicting the developmental potential of ovine embryos. The development of 37 long wool sheep embryos from IVF to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Image-Pro Plus software was then used to measure zona pellucida thickness, embryo diameter, cellular grey-scale pixel intensity and heterogeneity, and total area of the perivitelline space. A one-way analysis of variance (ANOVA) was done using SigmaPlot® 11.0 for all attributes at various time points during embryo development [i.e. presumptive zygote stage, t(0); first cleavage, t(2) or t(3); second cleavage, t(4) or t(6); and third cleavage, t(7) or t(8)]. Our results indicate that most parameters analysed did not differ among embryos varying in their developmental fate, with the exception of the perivitelline space area, which was greater (P&amp;lt;0.05) for non-dividing embryos than for future blastocysts at the presumptive zygote stage (4040±4137 vs. 857±642µm2, respectively; mean±s.d.). Consequently, the measurement of perivitelline space at t(0) could be used to predict developmental potential of invitro-produced ovine embryos, but further investigation is required.

Impact of sperm characteristics on time-lapse embryo morphokinetic parameters and clinical outcome of conventional in vitro fertilization.

Sperm abnormalities may negatively affect embryo development. To determine the influence of sperm abnormalities (morphology, motility, DNA fragmentation) on embryo morphokinetic variables and clinical outcome of conventional IVF. Participants were 86 couples undergoing in vitro fertilization (IVF). Sperm morphology was evaluated according to the strict criteria proposed by Kruger/Tygerberg. CASA system was applied for sperm motility assessment. Sperm DNA fragmentation was assessed by the chromatin structure assay (SCSA). Morphokinetic parameters were determined in 223 embryos obtained from conventional IVF only and cultured in a single-step medium using time-lapse imaging technology. Time-lapse variables from the initial embryo development, such as time of pronuclei fading (tPNf) and time for two cells (t2), were those more strongly related with abnormalities of sperm motility, morphology, and DNA fragmentation. Sperm morphological abnormalities rather than sperm motility were more closely associated with embryo morphokinetics. Sperm head defects were mainly correlated with the last stages of embryonic development (t9 to tHB), sperm midpiece defects with intermediate cleaving embryos (t5-t9), and sperm tail defects with the initial stages of embryonic development (tPNa-t4). Excess residual cytoplasm was positively correlated with all embryo morphokinetic parameters except t2 and tM. Absence of acrosomes, pinheads, coiled tails, and multiple sperm morphological defects correlated negatively with time-lapse embryo morphokinetic variables. A large number of sperm-related variables, including frequency of specific morphological defects, morphological indexes, DNA fragmentation and motility, and time-lapse embryo variables, such as time intervals based mainly of 15 time points were recorded. There were strong associations between specific sperm defects of the head, midpiece, and tail with certain stages of embryonic development from observation of pronuclei to the hatched blastocyst. Coiled tail, cumulative head defects, and multiple abnormalities index (MAI) were associated both with embryo morphokinetics and the implantation success.

Morphokinetic characteristics of embryos originating from extremely small follicles: A prospective study

ObjectiveTo investigate the developmental potential of oocytes and embryos derived from extremely small follicles (<10 mm) in comparison to those originated in larger follicles. Study designA prospective study, undertaken in a university affiliated single center tertiary hospital. The study included 98 patients undergoing infertility treatments. On the day of ovum pickup (OPU) follicles were counted and measured. Aspiration of follicles larger and smaller than 10 mm was undertaken separately and the development of embryos originating from oocytes from these follicles was followed up using different wells for each embryo. There was no low limit of size for aspiration. Each oocyte retrieved was marked for its origin and numbered for further follow up. We recorded: Oocytes retrieved, maturation stage, fertilization rate, cleavage rate, morphokinetic parameters, embryo transfers, embryo freezing, oocyte freezing and biopsy rate for preimplantation genetic diagnosis (PGD).Quality was evaluated by the morphokinetic parameters of the embryos developed using time-lapse imaging technology. Day 3 KIDScore was calculated to all embryos. ResultsSmall follicles compared to large follicles displayed lower recovery rate (45% vs. 74%, P < 0.0001), fewer matured oocytes (37.5% vs. 61.7%, P < 0.0001), higher rates of GV oocytes (20.7% vs., 3.7%, P < 0.0001), and lower fertilization rate (43.7% vs. 63.3%, P < 0.0001. However, morphokinetic variables were similar between embryos that originated from either small or large follicles. Median KIDscores were identical for embryos from small or large follicle origin. ConclusionsEmbryos originated from small follicles were not different than embryos from larger follicles, as assessed by morphokinetic parameters in time lapse system. In view of our findings, physicians should bear in mind that small follicle aspiration might yield good quality embryos.

G-1 Inhibits Breast Cancer Cell Growth via Targeting Colchicine-Binding Site of Tubulin to Interfere with Microtubule Assembly.

G-protein-coupled estrogen receptor 1 (GPER1) has been reported to play a significant role in mediating the rapid estrogen actions in a wide range of normal and cancer cells. G-1 was initially developed as a selective agonist for GPER. However, the molecular mechanisms underlying the actions of G-1 are unknown, and recent studies report inconsistent effects of G-1 on the growth of breast cancer cells. By employing high-resolution laser scanning confocal microscopy and time-lapse imaging technology, as well as biochemical analyses, in the current study, we provide convincing in vitro and in vivo evidence that G-1 is able to suppress the growth of breast cancer cells independent of the expression status of GPERs and classic estrogen receptors. Interestingly, we found that triple-negative breast cancer cells (TNBC) are very sensitive to G-1 treatment. We found that G-1 arrested the cell cycle in the prophase of mitosis, leading to caspase activation and apoptosis of breast cancer cells. Our mechanistic studies indicated that G-1, similar to colchicine and 2-methoxyestradiol, binds to colchicine binding site on tubulin, inhibiting tubulin polymerization and subsequent assembly of normal mitotic spindle apparatus during breast cancer cell mitosis. Therefore, G-1 is a novel microtubule-targeting agent and could be a promising anti-microtubule drug for breast cancer treatment, especially for TNBC treatment. Mol Cancer Ther; 16(6); 1080-91. ©2017 AACR.

A prospective randomized comparison of early embryo cleavage kinetics between two media culture systems.

Objective:To investigate whether early embryo cleavage kinetics were affected by type of culture media.Methods:In this prospective sibling-split study, 620 oocytes from 37 patients were randomly allocated into two groups: Cook group and Vitrolife group. Oocytes/embryos in Cook group, would be cultured with Cook sequential culture medium, while oocytes/embryos in Vitrolife group, would be cultured with Vitrolife sequential culture medium. Time-lapse imaging technology was used to calculate exact timing of early embryo cleavage events which included time to 2PN breakdown, cleavage to 2-, 3-, 4-, 5- cell and the time duration in the 2-,3-cell stage. Then these timing of early embryo cleavage events were compared between Cook group and Vitrolife group. Moreover, fertilization rate, cleavage rate, high quality embryo rate, usable blastocyst rate, pregnancy rate and implantation rate of these two groups were also analyzed.Results:The results showed there were no differences in all timing of early embryo cleavage events between the two groups. In addition, the two groups were similar in fertilization rate (Cook 71.0% vs. Vitrolife 71.3%, P>0.05), cleavage rate (Cook 98.1% vs. Vitrolife 98.2%, P>0.05), high quality embryo rate (Cook 52.1% vs. Vitrolife 52.7%, P>0.05), usable blastocyst rate (Cook 29.7% vs. Vitrolife 28.0%, P>0.05), pregnancy rate (Cook 46.7% VS. Vitrolife 50.0%, P>0.05) and implantation rate (Cook 30.3% VS. Vitrolife 29.0%, P>0.05).Conclusions:Morphokinetics used for embryo selection are not affected by the two different culture media.

Stem Cell Transplantation in Neuroscience: The Role of Molecular Imaging

The regenerative neurobiology research has become a great and interesting topic during the last years in basic but also in clinical neurosciences [3, 4]. This change has led to promising approaches in the development of stem cell transplantation and also to orchestrate endogenous repair mechanism not only in brain injuries and neurodegenerative diseases but more and more of other potential application possibilities in neurosciences [8, 9]. Such new knowledge creation is not only followed by a widening of therapeutic possibilities in different diseases of the field of neurosciences, but has also increased our understanding of (endogenous) molecular strategies to repair damaged brain [3]. Such research into cell transforming and reprogramming processes represent also a great step towards a personalized regeneration medicine in neurosciences by disclosing detailed reprogramming mechanisms [6]. However, stem cell biology remains still incompletely understood despite the described substantial advances in the field. Inefficient stem cell differentiation, difficulty in verifying successful delivery to the target organ, and problems with engraftment are only some of principal reasons that hamper the transition from laboratory animal studies to human clinical trials [8, 9]. In this context, it is important to shed light that “neuronal stem cell achieve their therapeutic efficacy exclusively by a cell-replacement mechanisms” or by “a milieu of (intrinsic) neuroprotective molecules, temporarly and spatially orchestrated by environmental needs (both cited from [1]). We have therefore to control and to better understand such stem cellmediated therapeutic effects: The current imaging methods are an excellent and state-of-the-art instrument for that purpose [6]. Molecular imaging is an instrument that has led to unprecedented progress in understanding the fundamental behavior of stem cells, including their survival, biodistribution, immunogenicity, and tumorigenicity in the targeted tissues of interest [3]. Both short-term and more permanent monitoring of stem cells in cultures and in live organisms have benefited from recently developed imaging approaches that are designed to investigate cell behavior and function. Confocal and multiphoton microscopy, time-lapse imaging technology, and series of noninvasive imaging technologies enable us to investigate cell behavior in the context of a live organism [6]. In turn, the knowledge gained has brought our understanding of stem cell biology to a new level. The increasing possibilities in molecular imaging as a diagnostic tool in the clinical neuroscience and its ability to provide baseline assessments in preclinical diseases models in neuroscience necessitate its implication in the translation of stem cell therapy into an efficient and successful treatment option in neuroscience [2]. The molecular imaging has brought a great and promising opportunity to neuroscience research: to study stem cell therapy in vivo [6, 8, 9]. Further strategies relying on reporter genes might in the near future provide interesting new possibilities to current strategies and broaden some of the current limitations [7, 9]. Current problems of molecular imaging in neurosciences are a low contrast and crossing of the BBB [2]. Even so molecular imaging in neurosciences gains increased interest for stem cell transplantation; it is often not adequately used. Molecular imaging is only one of the key instruments that should be used in such studies, but it should be combined with concomitant behavioral assessment of the patient [2]. Still in 2012, the main outcome measure of any therapeutic intervention must be the behavioral recovery of N. Sandu : T. Spiriev : B. Schaller (*) Department of Neurosurgery, University of Paris, Paris, France e-mail: bernhardjschaller@gmail.com

Molecular Imaging and Stem Cell Research

During the last decade, there has been enormous progress in understanding both multipotent stem cells such as hematopoietic stem cells and pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells. However, it has been challenging to study developmental potentials of these stem cells because they reside in complex cellular environments and aspects of their distribution, migration, engraftment, survival, proliferation, and differentiation often could not be sufficiently elucidated based on limited snapshot images of location or environment or molecular markers. Therefore, reliable imaging methods to monitor or track the fate of the stem cells are highly desirable. Both short-term and more permanent monitoring of stem cells in cultures and in live organisms have benefited from recently developed imaging approaches that are designed to investigate cell behavior and function. Confocal and multiphoton microscopy, time-lapse imaging technology, and series of noninvasive imaging technologies enable us to investigate cell behavior in the context of a live organism. In turn, the knowledge gained has brought our understanding of stem cell biology to a new level. In this review, we discuss the application of current imaging modalities for research of hematopoietic stem cells and pluripotent stem cells and the challenges ahead.

Puerarin attenuates glutamate-induced neurofilament axonal transport impairment

Puerarin (Pur) is a primary component of the most functional extracts of Pueraria lobata used in traditional Chinese medicine for centuries. Since it has been postulated that Pur protects the brain against glutamate (Glu) neurotoxicity, we investigated the effects of Pur on Glu-induced axonal transport impairment in primary hippocampal neurons in this study. Primary hippocampal cultures were prepared from 2-day-old Sprague-Dawley rats. Intracellular calcium concentration [Ca(2+)](i), neurofilament (NF) phosphorylation and protein kinase activity for Cdk5 were measured. Time-lapse imaging technology was used to capture the NF axonal transport in the cultured neurons with transiently transfected fluorescence protein linked to the N-terminus of NF-M (EGFP-NFM). The results showed that Pur significantly diminished the Glu-induced elevation of [Ca(2+)](i) in dose-dependent manner and antagonized the Glu-evoked increases in NF phosphorylation at protein levels. The neurons under the Glu treatment displayed the accumulation of immobile NF clusters in the cell body and the reduced rates of axonal transport of NFs by 72.8% compared to the control neurons. Intriguingly, Pur reversed the slowed rate of the axonal transport by 35.6%. Pur also remarkably attenuated Glu-evoked activation of Cdk5. Pur may play a role in protecting against Glu-induced NF axonal transport impairment in rat primary hippocampal neurons by inhibiting the increased [Ca(2+)](i) and by impeding the activation of Cdk5.

Increased expression of cdk5/p25 in N2a cells leads to hyperphosphorylation and impaired axonal transport of neurofilament proteins

Aims Alzheimer's disease (AD) is the leading cause of dementia. The increased cdk5 expression and enhanced phosphorylation of tau and NFs have been seen in AD patients. Our study aimed at investigating the effects of increased cdk5 activity on axonal transport of neurofilaments (NFs). Main methods In this study, we used a molecular engineering approach to overexpress cdk5/p25 in neuroblastoma N2a cells and investigated the effects on axonal transport with live cell imaging techniques. Key findings In stably transfected cells, there was a 2.5-fold increase in cdk5 activity compared to non-transfected cells, which in turn led to a dramatic increase in phosphorylation of NFs and tau at several phosphorylation sites. Using time-lapse imaging technology, the transport of NFs was captured in the cells overexpressing cdk5/p25, which were also transiently transfected with fluorescence protein linked to the N-terminus of NF-M (EGFP-NFM). The cdk5/p25 cells displayed significantly slower rates of axonal transport of NFs, with accumulation of immobile NF clusters observed in the cell body. Roscovitine, an inhibitor of cdk5, significantly reversed this defect in axonal transport. Significance These results suggest that increased cdk5 activity found in AD subjects may be crucially related to the pathogenesis of AD via an underlying mechanism by which it promotes accumulation of excessively phosphorylated cytoskeletal NF proteins, leading to the enduring impairment of axonal transport of NFs.