Label-free ghost cytometry for manufacturing of cell therapy products.

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However, current QC methods, reliant on molecular staining, pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC), a machine learning-driven, multidimensional, high-content, and high-throughput flow cytometry approach, in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified cell count and viability of human peripheral blood mononuclear cells (PBMCs) and identified non-apoptotic live cells and early apoptotic/dead cells in PBMCs (ROC-AUC: area under receiver operating characteristic curve = 0.975), T cells and non-T cells in white blood cells (ROC-AUC = 0.969), activated T cells and quiescent T cells in PBMCs (ROC-AUC = 0.990), and particulate impurities in PBMCs (ROC-AUC ≧ 0.998). The results support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers, assess viability, identify specific cell subsets or phenotypic states, and remove impurities during cell therapy manufacturing. Thus, LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency.

Pooled CRISPR screening of high-content cellular phenotypes using ghost cytometry.

Recent advancements in image-based pooled CRISPR screening have facilitated the mapping of diverse genotype-phenotype associations within mammalian cells. However, the rapid enrichment of cells based on morphological information continues to pose a challenge, constraining the capacity for large-scale gene perturbation screening across diverse high-content cellular phenotypes. In this study, we demonstrate the applicability of multimodal ghost cytometry-based cell sorting, including both fluorescent and label-free high-content phenotypes, for rapid pooled CRISPR screening within vast cell populations. Using the high-content cell sorter operating in fluorescence mode, we successfully executed kinase-specific CRISPR screening targeting genes influencing the nuclear translocation of RelA. Furthermore, using the multiparametric, label-free mode, we performed large-scale screening to identify genes involved in macrophage polarization. Notably, the label-free platform can enrich target phenotypes without requiring invasive staining, preserving untouched cells for downstream assays and expanding the potential for screening cellular phenotypes even when suitable markers are absent.

Label-free enrichment of human pluripotent stem cell-derived early retinal progenitor cells for cell-based regenerative therapies.

Pluripotent stem cell-based therapy for retinal degenerative diseases is a promising approach to restoring visual function. A clinical study using retinal organoid (RO) sheets was recently conducted in patients with retinitis pigmentosa. However, the graft preparation currently requires advanced skills to identify and excise suitable segments from the transplantable area of the limited number of suitable ROs. This remains a challenge for consistent clinical implementations. Herein, we enabled the enrichment of wild-type (non-reporter) retinal progenitor cells (RPCs) from dissociated ROs using a label-free ghost cytometry (LF-GC)-based sorting system, where a machine-based classifier was trained in advance with another RPC reporter line. The sorted cells reproducibly formed retinal spheroids large enough for transplantation and developed mature photoreceptors in the retinal degeneration rats. This method of enriching early RPCs with no specific surface antigens and without any reporters or chemical labeling is promising for robust preparation of graft tissues during cell-based therapy.

Label-free cell detection of acute leukemia using ghost cytometry.

Early diagnosis and prompt initiation of appropriate treatment are critical for improving the prognosis of acute leukemia. Acute leukemia is diagnosed by microscopic morphological examination of bone marrow smears and flow cytometric immunophenotyping of bone marrow cells stained with fluorophore-conjugated antibodies. However, these diagnostic processes require trained professionals and are time and resource-intensive. Here, we present a novel diagnostic approach using ghost cytometry, a recently developed high-content flow cytometric approach, which enables machine vision-based, stain-free, high-speed analysis of cells, leveraging their detailed morphological information. We demonstrate that ghost cytometry can detect leukemic cells from the bone marrow cells of patients diagnosed with acute lymphoblastic leukemia and acute myeloid leukemia without relying on biological staining. The approach presented here holds promise as a precise, simple, swift, and cost-effective diagnostic method for acute leukemia in clinical practice.

In silico-labeled ghost cytometry.

Characterization and isolation of a large population of cells are indispensable procedures in biological sciences. Flow cytometry is one of the standards that offers a method to characterize and isolate cells at high throughput. When performing flow cytometry, cells are molecularly stained with fluorescent labels to adopt biomolecular specificity which is essential for characterizing cells. However, molecular staining is costly and its chemical toxicity can cause side effects to the cells which becomes a critical issue when the cells are used downstream as medical products or for further analysis. Here, we introduce a high-throughput stain-free flow cytometry called in silico-labeled ghost cytometry which characterizes and sorts cells using machine-predicted labels. Instead of detecting molecular stains, we use machine learning to derive the molecular labels from compressive data obtained with diffractive and scattering imaging methods. By directly using the compressive 'imaging' data, our system can accurately assign the designated label to each cell in real time and perform sorting based on this judgment. With this method, we were able to distinguish different cell states, cell types derived from human induced pluripotent stem (iPS) cells, and subtypes of peripheral white blood cells using only stain-free modalities. Our method will find applications in cell manufacturing for regenerative medicine as well as in cell-based medical diagnostic assays in which fluorescence labeling of the cells is undesirable.

Lysine acetylation regulates the interaction between proteins and membranes.

Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.

Huntingtin fibrils with different toxicity, structure, and seeding potential can be interconverted.

The first exon of the huntingtin protein (HTTex1) important in Huntington's disease (HD) can form cross-ß fibrils of varying toxicity. We find that the difference between these fibrils is the degree of entanglement and dynamics of the C-terminal proline-rich domain (PRD) in a mechanism analogous to polyproline film formation. In contrast to fibril strains found for other cross-ß fibrils, these HTTex1 fibril types can be interconverted. This is because the structure of their polyQ fibril core remains unchanged. Further, we find that more toxic fibrils of low entanglement have higher affinities for protein interactors and are more effective seeds for recombinant HTTex1 and HTTex1 in cells. Together these data show how the structure of a framing sequence at the surface of a fibril can modulate seeding, protein-protein interactions, and thereby toxicity in neurodegenerative disease.

The Mitochondrial Peptide Humanin Targets but Does Not Denature Amyloid Oligomers in Type II Diabetes.

Mitochondrially derived peptides (MDPs) such as humanin (HN) have shown a remarkable ability to modulate neurological amyloids and apoptosis-associated proteins in cells and animal models. Recently, we found that humanin-like peptides also inhibit amyloid formation outside of neural environments in islet amyloid polypeptide (IAPP) fibrils and plaques, which are hallmarks of Type II diabetes. However, the biochemical basis for regulating amyloids through endogenous MDPs remains elusive. One hypothesis is that MDPs stabilize intermediate amyloid oligomers and discourage the formation of insoluble fibrils. To test this hypothesis, we carried out simulations and experiments to extract the dominant interactions between the S14G-HN mutant (HNG) and a diverse set of IAPP structures. Replica-exchange molecular dynamics suggests that MDPs cap the growth of amyloid oligomers. Simulations also indicate that HNG-IAPP heterodimers are 10 times more stable than IAPP homodimers, which explains the substoichiometric ability of HNG to inhibit amyloid growth. Despite this strong attraction, HNG does not denature IAPP. Instead, HNG binds IAPP near the disordered NFGAIL motif, wedging itself between amyloidogenic fragments. Shielding of NFGAIL-flanking fragments reduces the formation of parallel IAPP ß-sheets and subsequent nucleation of mature amyloid fibrils. From ThT spectroscopy and electron microscopy, we found that HNG does not deconstruct mature IAPP fibrils and oligomers, consistent with the simulations and our proposed hypothesis. Taken together, this work provides new mechanistic insight into how endogenous MDPs regulate pathological amyloid growth at the molecular level and in highly substoichiometric quantities, which can be exploited through peptidomimetics in diabetes or Alzheimer's disease.

The Mitochondrial-Derived Peptides, HumaninS14G and Small Humanin-like Peptide 2, Exhibit Chaperone-like Activity.

Mitochondrial-derived peptides (MDPs) and their analogs have emerged as wide-spectrum, stress response factors protective in amyloid disease models. MDP cytoprotective functions are generally attributed to anti-apoptotic activity, however, little is known about their capacity to facilitate the cell's unfolded protein response via direct interactions with amyloidogenic proteins. Here, we explored the effects of the MDP-analog, humaninS14G (HNG), and the MDP, small humanin-like peptide 2 (SHLP2), on the misfolding of islet amyloid polypeptide (IAPP), a critical pathogenic step in type 2 diabetes mellitus (T2DM). Our thioflavin T fluorescence studies show that HNG inhibits IAPP misfolding at highly substoichiometric concentrations. Seeded fluorescence and co-sedimentation studies demonstrate MDPs block amyloid seeding and directly bind misfolded, seeding-capable IAPP species. Furthermore, our electron paramagnetic resonance spectroscopy and circular dichroism data indicate MDPs do not act by binding IAPP monomers. Taken together our results reveal a novel chaperone-like activity wherein these MDPs specifically target misfolded amyloid seeds to inhibit IAPP misfolding which, along with direct anti-apoptotic activity and beneficial metabolic effects, make HNG and SHLP2 exciting prospects as T2DM therapeutics. These data also suggest that other mitochondrial stress response factors within the MDP family may be amenable to development into therapeutics for protein-misfolding diseases.

Diabetic Risk Factors Promote Islet Amyloid Polypeptide Misfolding by a Common, Membrane-mediated Mechanism.

The current diabetes epidemic is associated with a diverse set of risk factors including obesity and exposure to plastics. Notably, significant elevations of negatively charged amphiphilic molecules are observed in obesity (e.g. free fatty acids and phosphatidic acid) and plastics exposure (monophthalate esters). It remains unclear whether these factors share pathogenic mechanisms and whether links exist with islet amyloid polypeptide (IAPP) misfolding, a process central to ß-cell dysfunction and death. Using a combination of fluorescence, circular dichroism and electron microscopy, we show that phosphatidic acid, oleic acid, and the phthalate metabolite MBzP partition into neutral membranes and enhance IAPP misfolding. The elevation of negative charge density caused by the presence of the risk factor molecules stabilizes a common membrane-bound α-helical intermediate that, in turn, facilitates IAPP misfolding. This shared mechanism points to a critical role for the membrane-bound intermediate in disease pathogenesis, making it a potential target for therapeutic intervention.