1. A Single-Cell RNA Expression Map of Human Coronavirus Entry Factors (Singh et al., 2020; Cell Reports)

 

This article profiled an extensive range of coronaviruses, specifically coronavirus-associated receptors and factors (SCARFs) using single cell RNA-sequencing on a range of healthy human tissues.

 

Aside from the well-studied SCARFs such as ACE2 receptor and TMPRSSS2 protease which are crucial for coronaviruses such as SARS-CoV-2 to infect cells, the authors have found a total of 28 SCARFs that are potentially important in modulating viral entry to cells. They then analyzed the expression of these SCARFs in 10 different types of pluripotent and determined that pluripotent stem cells and/ or cells during very early stages of differentiation are unlikely to be permissive to SARS-CoV-2 infection.

 

One of the most interesting data shown in this paper is that there is a striking difference between the male and female reproduction organs in that during the early stages of spermatogenesis, cells are permissive to SARS-CoV-2 infection whilst the ovary and oocytes are unlikely to be infected.

 

After analyzing about 200,000 cells representing all major adult organs, the authors were able to determine that certain cell types express SCARFs such as enterocytes of small intestines, sinusoidal endothelium of liver etc. Although the nasal epithelium is the gateway to SARS-CoV-2 infection, the authors also found that the nasal epithelium also produces a high level of restriction factors against the viral infection.

 

This paper presents an in-depth look into the expression of SCARFs in various stages of development, different reproductive organs and different major organs in the body. This information is highly valuable in our efforts to understand how the SARS-CoV-2 infects and causes problems to not just the lungs but also to other vital organs. This information will also be valuable in the attempt to understand more about SARS-CoV-2 infection risk in a developing embryo and/or fetus.    

 

 2. Transplanted microvessels improve pluripotent stem cell-derived cardiomyocyte engraftment and cardiac function after infarction in rats (Sun et al., 2020; Sci Transl Med)

 

This interesting paper is published by the Nunes Lab at U Toronto in collaboration with Gordon Keller. This research article describes a method to increase the survival of stem cell-derived cardiomyocytes (heart cells) after transplantation into an infarcted heart which was co-transplanted the heart cells with ready-made microvessels obtained from adipose tissue.

 

The microvessels were obtained from adipose tissues where the tissues were digested with collagenase to dislodge cells such as stromal cells, adipocytes etc. It is incredible how these microvessels retained endothelial lining in the lumens and perivascular cell coverage. It is also amazing that these vessels have diameters ranging from 10-20um and are longer than 400uM in length.

 

The authors chose to perform the transplantation of the stem cell-derived cardiomyocytes subcutaneously in immunocompromised mice because it is a site of poor cardiomyocyte engraftment. However, when transplanted with the microvessels, there is a 6-fold increase in the cardiomyocyte survival post-transplantation and significant improvement in overall heart function, indicating that the vessels are aiding in in the vascularization of the cardiomyocyte graft.

 

This paper has important implications in improving stem cell-based transplantation therapy for the heart. In addition, obtaining functional and intact microvessels from adipose tissue then transplanting it with the cardiomyocytes is a highly innovative and potentially sustainable method.

 

3. Reinfection with SARS-CoV-2 and Failure of Humoral Immunity: a case report. (Goldman et al., 2020; MedRxiv)

 

** The paper was a pre-print article at the time this round-up was prepared**

 

This clinical paper reports an intriguing case of SARS-CoV-2 reinfection in a patient, which the second infection by the D614G mutated version of SARS-CoV-2.

 

This particular patient is a sexagenarian (Age between 60-69) and was diagnosed with COVID19 in early March. The patient then received treatment and supportive care for the symptoms they suffered. After 39 days, the patient was tested negative for COVID19. However, after 3.5 months, the patient returned to the ER and was tested positive for COVID19. However, interesting the patient was less severely ill as compared to March. This patient as treated with remdesivir (anti-viral drug) and dexamethasone (anti-inflammation drug).

 

Turns out, the patient was infected by a mutated strain of SARS-CoV-2 (D614G mutation; I made an infograph regarding the D614G mutated SARS-CoV-2), which is now known to have a higher replicative and infective capacity. The authors then perform extensive studies into the patient’s antibody responses as well as B-cell repertoires. They observed that the patients, although able to produce SARS-CoV-2 neutralizing antibodies within the first week of the first infection were unable to protect the patient from the D614G viral strain. In the B-cell repertoire, new B cell clones did not emerge after 18 days of reinfection, possibly suggesting a deficiency in developing a response to the new viral strain. Thankfully, the patient presented with milder symptoms after reinfection.

 

This case study is a highly valuable one, in that it will allow for better understanding about antibody responses towards SARS-CoV-2 after infection and also to better understand the risk of an re-infection by the more virulent D614G strain.

1. Centrosome Reduction Promotes Terminal Differentiation of Human Cardiomyocytes (Ng et al., 2020; Stem Cell Reports)

 

This is an interesting paper describing the role of centrosomes in the maturation of the heart cells, cardiomyocytes. Over the past decade, researchers have been working on unveiling the complexities of how the heart develops from a fetus to an adult and a large part of the work goes into understanding now cardiomyocytes mature from an active proliferating stage to a post-mitotic, non-dividing stage. Here, the authors determined that that centrosomes, the organelle that regulates cell cycle by organizing microtubules in the cells, are involved in cardiomyocyte maturation.

 

First, they target and inhibit centrosomes in stem cell-derived cardiomyocytes by using a small molecule called centrinone. They observed that the cardiomyocytes are less proliferative. With that, the cells also exhibited mature phenotypes such as hypertrophy (increase in cell size), increase expression of mature cardiac markers such as myosin heavy and light-chain isoforms as well as sarcomeric markers. The authors also replicated this observation in 3D cardiac organoids.

 

We (Yong et al., 2018) and others have shown that introducing CHIR99021, a GSK inhibitor, to differentiated cardiomyocytes can induce proliferation. Here, the authors observed that CHIR99021 treatment also induces centrosome return by increasing b-catenin localization to centrosomes.

 

Lastly, the authors show that Hippo kinase activation is important in promoting centrosome reduction in cardiomyocytes as they mature.

 

Although the paper did not thoroughly interrogate the maturity of the cardiomyocytes after the drug-induced centrosome loss, this study certainly still value-adds to the current pool of knowledge on cardiomyocyte maturation. This can also be valuable for others to look at how this method can be applied to obtain more mature cardiomyocytes in vitro for drug testing, disease modeling or even therapeutic purposes.

 

           

2. SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response (Huang et al., 2020; Cell Stem Cell)

 

**This was a pre-poof article when this round-ups were prepared and published.**

 

The authors here present a stem cell-based in vitro model to study SARS-CoV-2 infection. The authors used human pluripotent stem cells and differentiated them to the lung alveolar type 2 cells, which are basically lung epithelial cells, using a published protocol. As we know by now, a severe symptom associated with COVID19 is pneumonia as a result of infection and inflammation of the lung epithelial cells. Therefore, the authors aim to use stem cell-derived lung alveolar type 2 cells (iAT2s) to model and better understand the cellular events that occur after SARS-CoV-2 infection.

 

After confirming that iAT2s are able to be infected by SARS-CoV-2 in vitro, the authors performed bulk RNA-sequencing on the infected cells and showed an upregulation of NFKB signaling, a major inflammatory pathway and also a loss of mature alveolar cellular identities. Proliferation markers were also downregulated in infected iAT2s. This shift in cellular state is both striking and intriguing.

 

The authors also tested different drugs in their stem cell-based model such as the famous anti-viral drug Remdesivir and inhibitor of TMPRSS2 protease called Camostat. Both of which were able to reduce number of viral particles in the iAT2s.

 

The authors acknowledged that their study did not include lung alveolar type 1 cells (iAT1s) which are also lung epithelial cells that are affected during SARS-CoV-2 infection. Nonetheless, this paper is valuable in understanding SARS-CoV-2 infection in the iAT2s without the use of patients or post-mortem lung tissues.

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3.    Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR (Smyrlaki et al., 2020; Nature Communications)

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This is an interesting resource paper describing a shorter way to perform RT-PCR on samples during COVID19 diagnosis. Just to provide a quick background on the way COVID19 testing is done so this paper makes more sense – after the nasopharyngeal swab is done, the viral RNA (if any) is extracted from the swabs using RNA extraction kits that involves lysing (breaking apart) cells and viral particles to release RNA. Of course, the RNA is further processed and purified to make sure that they are free from contamination. Thereafter, the RNA is then converted into DNA (via a process called reverse transcription, RT) and the DNA samples are amplified via PCR. If the viral RNA is present in the samples, they will be amplified and detected during the PCR process.

 

You can imagine that the entire process is a lengthy one and requires a lot of molecular reagents and labour. Therefore, this group published their RNA extraction-free single reaction RT-PCR testing procedure. Instead of utilizes costly RNA kits, the authors suggest to collect swabs in a generic buffer that does not inhibit the RT-PCR process and then heated up or swabs can be collected directly into lysis buffer and the samples can be directly subjected to RT-PCR.

 

Overall, the authors have presented convincing evidence to support to use of heat-inactivation or detergent-based lysis in the COVID19 testing workflow. This method significantly reduces cost and time required for COVID19 testing allowing it to be more rapid and importantly, affordable.

1. Circadian Entrainment Triggers Maturation of Human In Vitro Islets (Alvarez-Dominguez et al., 2020; Cell Stem Cell)

 

This paper is from the renowned stem cell biologist + diabetes researcher Douglas Melton’s lab at Harvard. Here, they investigated into the epigenetics associated with differentiating human pluripotent stem cells (hPSCs) into pancreatic islets (i.e. the functional units of the pancreas). Using their lab’s published (and highly cited) differentiation protocol, they differentiated hPSCs into pancreatic islets that compose of pancreatic b-cells (cells that secrete insulin) and other cell types including polyhormonal cells (secretes both insulin and glucagon). During the course of differentiation, they isolated cells at all stages of differentiation such as definitive endoderm cells, early and late pancreatic progenitors, endocrine progenitors as well as the pancreatic b-cells and polyhormonal cells.

 

The lab utilized an extensive list of epigenetic assays such as whole-genome bisulfite sequencing (WGBS), assay for transposase-accessible chromatin by sequencing (ATAC-seq), chromatin immunoprecipitation sequencing for two histone marks (H3K27ac and H3K4me1) and also RNA-sequencing. Overall, they managed to show drastic epigenetic switches upon endocrine commitment. For instance, hypermethylation is highest upon pluripotency exit (reflecting chromatin silencing) but hypomethylation is prevalent during pancreatic specification (reflecting chromatin activation). They also found that the epigenome of pancreatic b-cells are already primed towards beta-cell fate while the polyhormonal cells are actually primed towards alpha-cell fate.

 

When the group was investigating into the core regulatory circuits across islet differentiation and maturation, they reported on the involvement of transcription factor LMX1B (novel discovery) in regulating the endocrine progenitor stage of differentiation. Knocking out LMX1B impairs islet differentiation.

 

What is really interesting is that they also found that several circadian factors were involved in the regulation of pancreatic beta-cell stage of differentiation. Circadian cycle is also known as the body’s internal clock which responds to night and day, sleep and wake. The authors exposed their differentiated islets to a cycle of feeding and fasting (using stimuli such as glucose, arginine, forskolin and insulin) promoted increased in insulin secretion by 6-fold. This in vitro feeding and fasting is known as clock-entrainment. In addition, the authors performed RNA-sequencing on clock-entrained islets and detected more than 10,000 genes that oscillate with the feeding and fasting cycles. Genes involved in metabolism and islet maturation were induced in the clock-entrained islets.

 

Lastly, the authors transplanted clock-entrained islets into immunocompromised mice and observed that they showed higher glucose-induced insulin secretion as early as 3 days post-transplantation.

 

In conclusion, the authors did a comprehensive study into the epigenetics of islet differentiation. They also uncovered novel epigenetic and non-epigenetic regulators of differentiation. Most interestingly, they found that performing clock entrainment on their differentiated islets improved maturity and functionality. This study provided in-depth and novel insights into islet differentiation and essentially, will help the field in striving towards getting better quality pancreatic islets for transplantation purposes.

 

 

2. Human Mesenchymal Stromal Cells are resistant to SARS-CoV-2 Infection under Steady State, Inflammatory Conditions and in the Presence of SARS-CoV-2 infected Cells (Schäfer et al., 2020; Stem Cell Reports)

 

This is an interesting COVID19 paper (pre-proof) that showed that Mesenchymal Stromal Cells aka Mesenchymal Stem Cells (MSCs) are resistant to SARS-CoV-2 infection due to low expression of ACE2 (receptor that the virus recognizes and binds to) and TMPRSS2 (serine protease that is needed for viral entry into host cells). Even under inflammatory conditions, ACE2 and TMPRSS2 expression remains low and inflamed MSCs remained un-infected.

 

MSCs produce IDO-1, a metabolic enzyme which catabolizes Tryptophan, in response to inflammatory responses. This process is known to exert immunosuppressive effects. The authors confirmed that in the presence of SARS-CoV-2, the ability of MSCs in producing IDO-1 was not impaired.

 

Overally, this has important implication in the use of MSCs to treat lung inflammation which is a common and often severe symptom of COVID19. This study might suggest that transplanted MSCs into patients of COVID19 might not get infected by the viruses that are already circulating the patients and can thereby act to ameliorate inflammation.

 

 

3. Cell-Based Strain Remodeling of a Nonfibrous Matrix as an Organizing Principle for Vasculogenesis (Rüdiger et al., 2020; Cell Reports)

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This paper describes the underlying mechanism of how endothelial cells (ECs) are able to form vascular networks on a basement membrane such as Matrigel in vitro. When cells are seeded onto Matrigel, there is a ‘finding phase’ where ECs detect other ECs before moving and reorganizing themselves into tubes. This process is unsurprisingly dependent on cell density i.e. only at a threshold of cell densities will ECs be able to form networks of tubes.

 

So how do ECs communicate and ‘find’ one another? Surprisingly, the authors found that when ECs were depleted of growth factors or exposed to vascular endothelial growth factor (VEGF), there are no differences in tube formation capacities. It seems like protein gradients are not responsible for the process. Turns out, ECs are able to deform and restructure the basement matrices to do so. They essentially remodel the basement matrices to form matrix bridges between themselves and the cells that are arranged in tubes sit on top of the remodeled matrix bridges. Intriguingly, when the cells are removed from the matrix, the bridges persistent for at least 20h and when new ECs are re-seeding onto the de-cellularized matrix bridges, they rapidly aligned to the previous patents indicating that the matrix bridges are sufficient cues for tube formation.

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There are two main drivers of this phenomenon. First, the authors found that laminin, specifically the cross-linking of laminin in the Matrigel is needed for the tube formation process to occur. Second, it seems like the matrix bridges resulted in the creation of stiff regions on the matrix. It is likely that ECs utilized this stiffness cue to guide them in their tube formation.

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Overall, this paper serves to better understand how ECs form tubes in vitro on Matrigel which could potentially provide insights into the underlying mechanisms of vaculogenesis and angiogenesis in the body. Not only so, this could also provide knowledge on how cells remodel the basement matrix in the context of tissue fibrosis.