SARS-CoV-2 Infects Red Blood Cell Progenitors and Dysregulates Hemoglobin and Iron Metabolism.

BACKGROUND: SARS-CoV-2 infection causes acute respiratory distress, which may progress to multiorgan failure and death. Severe COVID-19 disease is accompanied by reduced erythrocyte turnover, low hemoglobin levels along with increased total bilirubin and ferritin serum concentrations. Moreover, expansion of erythroid progenitors in peripheral blood together with hypoxia, anemia, and coagulopathies highly correlates with severity and mortality. We demonstrate that SARS-CoV-2 directly infects erythroid precursor cells, impairs hemoglobin homeostasis and aggravates COVID-19 disease. METHODS: Erythroid precursor cells derived from peripheral CD34+ blood stem cells of healthy donors were infected in vitro with SARS-CoV-2 alpha variant and differentiated into red blood cells (RBCs). Hemoglobin and iron metabolism in hospitalized COVID-19 patients and controls were analyzed in plasma-depleted whole blood samples. Raman trapping spectroscopy rapidly identified diseased cells. RESULTS: RBC precursors express ACE2 receptor and CD147 at day 5 of differentiation, which makes them susceptible to SARS-CoV-2 infection. qPCR analysis of differentiated RBCs revealed increased HAMP mRNA expression levels, encoding for hepcidin, which inhibits iron uptake. COVID-19 patients showed impaired hemoglobin biosynthesis, enhanced formation of zinc-protoporphyrine IX, heme-CO2, and CO-hemoglobin as well as degradation of Fe-heme. Moreover, significant iron dysmetablolism with high serum ferritin and low serum iron and transferrin levels occurred, explaining disturbances of oxygen-binding capacity in severely ill COVID-19 patients. CONCLUSIONS: Our data identify RBC precursors as a direct target of SARS-CoV-2 and suggest that SARS-CoV-2 induced dysregulation in hemoglobin- and iron-metabolism contributes to the severe systemic course of COVID-19. This opens the door for new diagnostic and therapeutic strategies.

Raman Trapping Microscopy for Non-invasive Analysis of Biological Samples.

Raman microscopy is an emerging tool in biomedicine. It provides label-free and non-invasive analysis of biological cells. Due to its high biochemical specificity, Raman spectroscopy can be used to acquire spectral fingerprints that allow characterizing cells types and states. Here, we present a methodological approach for implementing Raman microscopy in skin cell measurements. Raman spectra can clearly identify keratinocytes, fibroblasts, and melanocytes cells that are involved in the production of autologous skin grafts. Consequently, Raman microscopy is a promising tool that can be used to analyze single cells and to test the quality of therapeutic cell products.

Cellular mechanisms of hereditary photoreceptor degeneration - Focus on cGMP.

The cellular mechanisms underlying hereditary photoreceptor degeneration are still poorly understood, a problem that is exacerbated by the enormous genetic heterogeneity of this disease group. However, the last decade has yielded a wealth of new knowledge on degenerative pathways and their diversity. Notably, a central role of cGMP-signalling has surfaced for photoreceptor cell death triggered by a subset of disease-causing mutations. In this review, we examine key aspects relevant for photoreceptor degeneration of hereditary origin. The topics covered include energy metabolism, epigenetics, protein quality control, as well as cGMP- and Ca2+-signalling, and how the related molecular and metabolic processes may trigger photoreceptor demise. We compare and integrate evidence on different cell death mechanisms that have been associated with photoreceptor degeneration, including apoptosis, necrosis, necroptosis, and PARthanatos. A special focus is then put on the mechanisms of cGMP-dependent cell death and how exceedingly high photoreceptor cGMP levels may cause activation of Ca2+-dependent calpain-type proteases, histone deacetylases and poly-ADP-ribose polymerase. An evaluation of the available literature reveals that a large group of patients suffering from hereditary photoreceptor degeneration carry mutations that are likely to trigger cGMP-dependent cell death, making this pathway a prime target for future therapy development. Finally, an outlook is given into technological and methodological developments that will with time likely contribute to a comprehensive overview over the entire metabolic complexity of photoreceptor cell death. Building on such developments, new imaging technology and novel biomarkers may be used to develop clinical test strategies, that fully consider the genetic heterogeneity of hereditary retinal degenerations, in order to facilitate clinical testing of novel treatment approaches.

Rapid Analysis of Cell-Nanoparticle Interactions using Single-Cell Raman Trapping Microscopy.

Iron oxide nanoparticles have been used in preclinical studies to label stem cells for non-invasive tracking and homing. The search continues for novel particle candidates that are suitable for clinical applications. Since standard analyses to investigate cell-particle interactions and safety are labor-intensive, an efficient procedure is required to guide future particle development and to exclude adverse health effects. The application of combined Raman trapping microscopy with fluidic chips is reported for the analysis of single cells labeled with different types of aminated iron oxide particles. Multivariate data analysis revealed Raman signal differences that could be clearly assigned to cell-particle interactions and cytotoxicity, respectively. A validation dataset verified that more than 95 % of the spectra were correctly classified. Thus, our approach enables rapid discrimination of non-hazardous from cytotoxic nanoparticles as a prerequisite for safe clinical applications.

Potential and limitations of microscopy and Raman spectroscopy for live-cell analysis of 3D cell cultures.

Today highly complex 3D cell culture formats that closely mimic the in vivo situation are increasingly available. Despite their wide use, the development of analytical methods and tools that can work within the depth of 3D-tissue constructs lags behind. In order to get the most information from a 3D cell sample, adequate and reliable assays are required. However, the majority of tools and methods used today have been originally designed for 2D cell cultures and translation to a 3D environment is in general not trivial. Ideally, an analytical method should be non-invasive and allow for repeated observation of living cells in order to detect dynamic changes in individual cells within the 3D cell culture. Although well-established laser confocal microscopy can be used for these purposes, this technique has serious limitations including penetration depth and availability. Focusing on two relevant analytical methods for live-cell monitoring, we discuss the current challenges of analyzing living 3D samples: microscopy, which is the most widely used technology to observe and examine cell cultures, has been successfully adapted for 3D samples by recording of so-called "z-stacks". However the required equipment is generally very expensive and therefore access is often limited. Consequently alternative and less advanced approaches are often applied that cannot capture the full structural complexity of a 3D sample. Similarly, image analysis tools for quantification of microscopic images range from highly specialized and costly to simplified and inexpensive. Depending on the actual sample composition and scientific question the best approach needs to be assessed individually. Another more recently introduced technology for non-invasive cell analysis is Raman micro-spectroscopy. It enables label-free identification of cellular metabolic changes with high sensitivity and has already been successful applied to 2D and 3D cell cultures. However, its future significance for cell analysis will strongly depend on the availability of application oriented and user-friendly systems including specific tools for easy analysis and interpretation of spectral data focusing on biological relevant information.

An engineered 3D human airway mucosa model based on an SIS scaffold.

To investigate interrelations of human obligate airway pathogens, such as Bordetella pertussis, and their hosts test systems with high in vitro/in vivo correlation are of urgent need. Using a tissue engineering approach, we generated a 3D test system of the airway mucosa with human tracheobronchial epithelial cells (hTEC) and fibroblasts seeded on a clinically implemented biological scaffold. To investigate if hTEC display tumour-specific characteristics we analysed Raman spectra of hTEC and the adenocarcinoma cell line Calu-3. To establish optimal conditions for infection studies, we treated human native airway mucosa segments with B. pertussis. Samples were processed for morphologic analysis. Whereas our test system consisting of differentiated epithelial cells and migrating fibroblasts shows high in vitro/in vivo correlation, hTEC seeded on the scaffold as monocultures did not resemble the in vivo situation. Differences in Raman spectra of hTEC and Calu-3 were identified in distinct wave number ranges between 720 and 1662 cm(-1) indicating that hTEC do not display tumour-specific characteristics. Infection of native tissue with B. pertussis led to cytoplasmic vacuoles, damaged mitochondria and destroyed epithelial cells. Our test system is suitable for infection studies with human obligate airway pathogens by mimicking the physiological microenvironment of the human airway mucosa.

Stem cell metabolic and spectroscopic profiling.

Stem cells offer great potential for regenerative medicine because they regenerate damaged tissue by cell replacement and/or by stimulating endogenous repair mechanisms. Although stem cells are defined by their functional properties, such as the potential to proliferate, to self-renew, and to differentiate into specific cell types, their identification based on the expression of specific markers remains vague. Here, profiles of stem cell metabolism might highlight stem cell function more than the expression of single genes/markers. Thus, systematic approaches including spectroscopy might yield insight into stem cell function, identity, and stemness. We review the findings gained by means of metabolic and spectroscopic profiling methodologies, for example, nuclear magnetic resonance spectroscopy (NMRS), mass spectrometry (MS), and Raman spectroscopy (RS), with a focus on neural stem cells and neurogenesis.

A compartment-specific transcriptome analysis reveals survival-relevant marker genes in the stroma fraction of squamous non-small cell lung cancer.

Although it is increasingly recognized that the tumor biology is influenced by the tumor stroma, prognostic gene signatures are usually derived from tissue consisting of tumor cells and surrounding stroma. This study presents a compartment-specific transcriptome analysis of lung squamous cell carcinoma (SCC) samples microdissected into tumor parenchyma and stroma fractions. Typical tumor and stroma genes were identified based on the expression ratios between the two compartments. Our results indicate that in SCC many markers related to longer survival are predominantly expressed in the stroma, particularly genes of the MHC-II complex. Stromal upregulation of MHC-II genes seems crucial for a clinically relevant antitumor immune response in SCC.

Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy.

For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds.

Laser-assisted selection and passaging of human pluripotent stem cell colonies.

The derivation of somatic cell products from human embryonic stem cells (hESCs) requires a highly standardized production process with sufficient throughput. To date, the most common technique for hESC passaging is the manual dissection of colonies, which is a gentle, but laborious and time-consuming process and is consequently inappropriate for standardized maintenance of hESC. Here, we present a laser-based technique for the contact-free dissection and isolation of living hESCs (laser microdissection and pressure catapulting, LMPC). Following LMPC treatment, 80.6+/-8.7% of the cells remained viable as compared to 88.6+/-1.7% of manually dissected hESCs. Furthermore, there was no significant difference in the expression of pluripotency-associated markers when compared to the control. Flow cytometry revealed that 83.8+/-4.1% of hESCs isolated by LMPC expressed the surface marker Tra-1-60 (control: 83.9+/-3.6%). In vitro differentiation potential of LMPC treated hESCs as determined by embryoid body formation and multi-germlayer formation was not impaired. Moreover, we could not detect any overt karyotype alterations as a result of the LMPC process. Our data demonstrate the feasibility of standardized laser-based passaging of hESC cultures. This technology should facilitate both colony selection and maintenance culture of pluripotent stem cells.

Noncontact laser microdissection and pressure catapulting: sample preparation for genomic, transcriptomic, and proteomic analysis.

The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitates accurate identification, isolation, and finally characterization of a specific cell or a population of cells and subsequently their subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within a single experiment, as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin is required. Sample preparation is therefore a very crucial step for high-resolution downstream applications. Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homogeneous sample preparation, resulting in an eminent increase in the specificity of molecular analyses. For microdissection, the force of focused laser light is used to excise selected cells or large tissue areas from object slides or from living cell culture down to a resolution of individual single cells and subcellular components like organelles or chromosomes, respectively. After microdissection this sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Wherever homogenous samples are required for subsequent analysis of, e.g., cell areas, single cells, or chromosomes, the PALM MicroBeam system is an indispensable tool. The integration of image analysis platforms fully automates screening, identification, and finally subsequent high-throughput sample handling. These samples can be directly linked into versatile downstream applications, such as single-cell mRNA-extraction, different PCR methods, microarray techniques, and many others. Acceleration in sample generation vastly increases the throughput in molecular laboratories and leads to an increasing knowledge about differentially regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and therefore enabling the development of systems for tumor biomarker identification, early detection of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.

Live cell catapulting and recultivation.

Laser micromanipulation systems are used worldwide in the field of life science research. Most of their applications focus on the isolation of specific cells from different types of tissue and the manipulation of subcellular structures within fixed or living cells. Using the PALM MicroBeam, it is possible to microdissect living cells from a cell culture, to catapult them into collection devices, and to re-cultivate the isolated cells. For this purpose, new protocols and special equipment were developed. It has also been demonstrated that Laser Microdissection and Pressure Catapulting (LMPC) have no influence on the proliferation rate of the cells. Even re-cultivated cell colonies, trypsinized and seeded out again, are still viable after a second LMPC-procedure. This new approach opens a wide field of interesting applications in cell biology, molecular pathology, and pharmacology.

High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology.

Isolation of intact RNA in high quality is the first and often the most critical step in performing many fundamental molecular biology experiments, and is essential for many techniques used in gene expression analysis. As many factors influence nucleic acid preservation, RNA isolation should include some important steps before and after the actual RNA extraction. We tested the influence of fixation and staining protocols regarding RNA integrity and concentration. A factor that is often underestimated is the absolute necessity for homogenous starting materials. Application of the LMPC technology allows for a rapidand highly precise procurement of purified cell populations suitable for a variety of downstream analyses.

Significance of loss of heterozygosity of the RB1 gene during tumour progression in well-differentiated liposarcomas.

Tumour progression can be investigated in liposarcomas showing a transition from a low-grade well-differentiated (WD) to a high-grade dedifferentiated (DD) variant. As RB1 gene alterations are common defects in sarcomas, this study examined the frequency of RB1 loss of heterozygosity (LOH) in a group of 14 well-differentiated liposarcomas (WDLs) and 17 well-differentiated/dedifferentiated liposarcomas (WD/DDLs), using a microdissection approach (PALM laser pressure catapulting) that allows the two histological components to be separated for polymerase chain reaction (PCR) analysis. In addition, RB1 protein expression and the Mib1 proliferation index were determined by immunohistochemistry and interphase FISH was performed with an RB1 probe at 13q14. By the use of four intragenic polymorphic RB1 markers (introns 1, 17, 20, and 25) for PCR, allelic losses were found only in the DD parts, but never in the pure WDLs or in the WD components of the WD/DDLs investigated. Furthermore, DD areas characterized by a heterogeneous RB1 protein expression pattern (35-65% immunopositivity), as compared with 90-100% RB1 positivity in WD areas, showed a marked increase in Mib1 proliferation index (19.6% versus 1.8% in WD areas; p<0.001). Interphase fluorescence in situ hybridization (FISH) detected a higher RB1-LOH rate in the DD components of WD/DDLs. Considering the different detection sensitivities of the three methodologies, it is concluded that loss of RB1 function already begins in the WDL, and that the tumour cell population with RB1-LOH starts prevailing in the tumour mass during progression of a WDL.

Analysis of microdissected prostate tissue with ProteinChip arrays--a way to new insights into carcinogenesis and to diagnostic tools.

Prostate carcinomas are one of the most common malignancies in western societies. The pathogenesis of this tumor is still poorly understood. These tumors present with two characteristic features: epithelial-mesenchymal interactions, which play a pivotal role for tumor development and most of clinically manifest cancers arise in prostate proper compared to a minority of tumors which develop in the transitional zone. Deciphering the epithelial-mesenchymal cross talk and identification of molecular pecularities of the sub-populations of cells in different zones can therefore help understanding carcinogenesis and development of new, non-invasive tools for the diagnosis and prognosis of prostate carcinomas which has remained a challenge until today. A ProteinChip array technology (SELDI = surface enhanced laser desorption ionization) has been developed recently by Ciphergen Biosystems enabling analysis and profiling of complex protein mixtures from a few cells. This study describes the analysis of approximately 500-1000 freshly obtained prostate cells by SELDI-TOF-MS (surface enhanced laser desorption ionization time-of-flight mass spectrometry). Pure cell populations of stroma, epithelium and tumor cells were selected by laser assisted microdissection. Multiple specific protein patterns were reproducibly detected in the range from 1.5 to 30 kDa in 28 sub-populations of 4 tumorous prostates and 1 control. A specific 4.3 kDa peak was increased in the prostate tumor stroma compared to normal prostate proper and transitional zone stroma and increased in prostate tumor glands compared to normal prostate proper and transitional zone glands. Coupling laser assisted microdissection with SELDI provides tremendous opportunities to identify cell and tumor specific proteins to understand molecular events underlying prostate carcinoma development. It underlines the vast potential of this technology to better understand pathogenesis and identify potential candidates for new specific biomarkers in general which could help to screen for and distinguish disease entities, i.e. between clinically significant and insignificant carcinomas of the prostate.