Machine learning aided single cell image analysis improves understanding of morphometric heterogeneity of human mesenchymal stem cells.

The multipotent stem cells of our body have been largely harnessed in biotherapeutics. However, as they are derived from multiple anatomical sources, from different tissues, human mesenchymal stem cells (hMSCs) are a heterogeneous population showing ambiguity in their in vitro behavior. Intra-clonal population heterogeneity has also been identified and pre-clinical mechanistic studies suggest that these cumulatively depreciate the therapeutic effects of hMSC transplantation. Although various biomarkers identify these specific stem cell populations, recent artificial intelligence-based methods have capitalized on the cellular morphologies of hMSCs, opening a new approach to understand their attributes. A robust and rapid platform is required to accommodate and eliminate the heterogeneity observed in the cell population, to standardize the quality of hMSC therapeutics globally. Here, we report our primary findings of morphological heterogeneity observed within and across two sources of hMSCs namely, stem cells from human exfoliated deciduous teeth (SHEDs) and human Wharton jelly mesenchymal stem cells (hWJ MSCs), using real-time single-cell images generated on immunophenotyping by imaging flow cytometry (IFC). We used the ImageJ software for identification and comparison between the two types of hMSCs using statistically significant morphometric descriptors that are biologically relevant. To expand on these insights, we have further applied deep learning methods and successfully report the development of a Convolutional Neural Network-based image classifier. In our research, we introduced a machine learning methodology to streamline the entire procedure, utilizing convolutional neural networks and transfer learning for binary classification, achieving an accuracy rate of 97.54%. We have also critically discussed the challenges, comparisons between solutions and future directions of machine learning in hMSC classification in biotherapeutics.

Guidelines for establishing a cytometry laboratory.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth.

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity. This warrants methodologies to provide purified, homogeneous populations of MSCs for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify stem cells from human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the "bright," positive-expressors of MSCs using multiparametric flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages. The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

MSCProfiler: a single cell image processing workflow to investigate mesenchymal stem cell heterogeneity.

Single cell cytometry has demonstrated plausible immuno-heterogeneity of mesenchymal stem cells (MSCs) owing to their multivariate stromal origin. To contribute successfully to next-generation stem cell therapeutics, a deeper understanding of their cellular morphology and immunophenotype is important. In this study, the authors describe MSCProfiler, an image analysis pipeline developed using CellProfiler software. This workflow can extract geometrical and texture features such as shape, size, eccentricity and entropy, along with intensity values of the surface markers from multiple single cell images obtained using imaging flow cytometry. This screening pipeline can be used to analyze geometrical and texture features of all types of MSCs across different passages hallmarked by enhanced feature extraction potential from brightfield and fluorescent images of the cells.

Polycaprolactone/graphene oxide/acellular matrix nanofibrous scaffolds with antioxidant and promyelinating features for the treatment of peripheral demyelinating diseases.

Peripheral demyelinating diseases entail damage to axons and Schwann cells in the peripheral nervous system. Because of poor prognosis and lack of a cure, this group of diseases has a global impact. The primary underlying cause of these diseases involves the inability of Schwann cells to remyelinate the damaged insulating myelin around axons, resulting in neuronal death over time. In the past decade, extensive research has been directed in the direction of Schwann cells focusing on their physiological and neuroprotective effects on the neurons in the peripheral nervous system. One cause of dysregulation in the remyelinating function of Schwann cells has been associated with oxidative stress. Tissue-engineered biodegradable scaffolds that can stimulate remyelination response in Schwann cells have been proposed as a potential treatment strategy for peripheral demyelinating diseases. However, strategies developed to date primarily focussed on either remyelination or oxidative stress in isolation. Here, we have developed a multifunctional nanofibrous scaffold with material and biochemical cues to tackle both remyelination and oxidative stress in one matrix. We developed a nanofibrous scaffold using polycaprolactone (PCL) as a foundation loaded with antioxidant graphene oxide (GO) and coated this bioscaffold with Schwann cell acellular matrix. In vitro studies revealed both antioxidant and remyelination properties of the developed bioscaffold. Based on the results, the developed multifunctional bioscaffold approach can be a promising biomaterial approach for treating demyelinating diseases.

Mammalian Chromosome Analysis and Sorting by Flow Cytometry.

The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mitotic block and cell harvesting Basic Protocol 2: Propidium iodide isolation Support Protocol 1: Swelling test Basic Protocol 3: MgSO4 low-molecular-weight isolation Basic Protocol 4: Polyamine high-molecular-weight isolation Support Protocol 2: Molecular-weight determination of chromosomal DNA Basic Protocol 5: Chromosome analysis and sorting.

Impact of process variations on splitter-tree-based integrated optical phased arrays.

We consider the impact of intra-wafer systematic spatial variation, pattern density mismatch, and line edge roughness on splitter-tree-based integrated optical phased arrays. These variations can substantially affect the emitted beam profile in the array dimension. We study the effect on different architecture parameters, and the analysis is shown to be consistent with experimental results.

Solutions for Shared Resource Lab Remote Quality Control and Instrument Troubleshooting during a Pandemic.

The COVID-19 pandemic has dramatically affected shared resource lab (SRL) staff in-person availability at institutions globally. This article discusses the challenges of ensuring reliable instrument performance and quality data output while facility staff and external service provider on-site presence is severely limited. Solutions revolve around the adoption of remote monitoring and troubleshooting platforms, provision of self-service troubleshooting resources specific to facility instruments and workflows, development of an assistance contact policy, and ensuring efficiency of limited in-person staff time. These solutions employ software and hardware tools that are already in use or readily available in the SRL community, such as remote instrument access tools, video hosting and conferencing platforms, and ISAC shared resources. © 2020 International Society for Advancement of Cytometry.

A stable hybrid containing haploid genomes of two obligate diploid Candida species.

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.