1. Stem cell-derived polarized hepatocytes (Dao Thi et al., 2020; Nature Commun)

 

I presented on this paper during a recent journal club and I thoroughly enjoyed it. This group described a method to generate columnar polarized hepatocyte-like cells (HLCs) from human pluripotent cells. These polarized hepatocytes are shown to have clearly defined basolateral and apical poles, separated by tight junctions. The technique is relatively straightforward  – first they differentiate hPSCs to the definitive endoderm lineage for 5 days on a typical 2-dimensional cell culture plate. They then seed the cells onto a transwell filter, which is a permeable little well that can be inserted into regular tissue culture plates. That way, the ‘top’ and the ‘bottom’ of the transwells are separated physically, allowing them to add different medias on the ‘top’ and ‘bottom’. Using this method, the HLCs were able to undergo hepatic multi-polar polarization and form distinct bile caniculi.

​

I especially love the second figure of the paper where they used transmission electron microscopy to show that that polarized HLCs had significantly better and healthier cellular organization than non-polarized ones. They also stained the cells with different polarization markers and did 3D imaging to show that the expression of the polarization markers were confined to their respective poles i.e. apical/ basal poles. Non-polarized HLCs, in contrast, did not have a clear distribution of those markers. Essentially, the transwell promoted polarization, with the apical pole facing the ‘top’ and basal facing the ‘bottom’.

​

Next the group was thorough in showing that the bile caniculi formed by these polarized HLCs were functional, can be infected with viruses and metabolize drugs; indicating that it potentially is a more superior platform to study liver physiology, drug testing and even virology.

 

 2. A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and Their Differentiated Derivatives (El-Nachef et al., 2020; Stem Cell Reports)

 

This is more of a resource paper where the authors from University of Washington generated a rainbow iPSC reporter. Those that are familiar with the rainbow transgenic mouse/ confetti mouse would understand why this is such an exciting news. In brief, a rainbow mouse is genetically engineered such that a single cell expresses a single fluorescent protein i.e. colour, therefore cell colonies arising from that one cells expresses the same colour. So, the rainbow iPSC reporter works essentially in the same way. Well, what is the use of a rainbow mouse in the first place? A rainbow mouse is especially important in the field of developmental biology where knowledge on how individual cells proliferation, expand and migrate are really important. Having a colour-labelled cells will allow us to track each cell and know what they are up to during development.

 

Here, the authors described their process of generating and validating the cell line, making sure that the cells were still pluripotent and were able to differentiate to all germ layers. The authors then differentiated the iPSC reporter to cortical neurons and they showed that a few clonally dominant neural progenitor cells generated most of the cortical neurons in the culture, which is super interesting (biological relevance is unclear). This information would otherwise be not known if it was just differentiating regular iPSCs to cortical neurons.

 

This is just one example of the potential uses of the rainbow iPSC reporter cell line. This resource is definitely a valuable one and has multiple uses across different fields like development, disease, drug discover, cell therapies etc.

 

3.     Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells (Sarkar et al., 2020; Nature Commun)

​

This is a really interesting paper from Vittorio Sebastiano’s lab here in Stanford. There is nothing like a paper that shows the rejuvenation of aged cells, essentially reverting aging. Here, they obtained fibroblasts and endothelial cells from aged human subjects and introduce a cocktail of mRNAs OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG ( OSKMLN) to the cells as part of a transient reprogramming strategy. They transfected OSKMLN to the cells for 4 consecutive days and saw that the gene expression of the reprogrammed cells were more similar to young cells as compared to aged cells. What is most surprising is that the cells retained their cellular identity and did not express other pluripotency markers other than the ones that were overexpressed from the transfection. It seems like OSKMLN promoted the activation of a ‘more youthful gene profile’. In addition, they also showed that OSKMLN significantly reverted the epigenetic clock of the aged cells.

 

The authors did a very thorough job in analyzing other aspects of physiological aging in vitro such as formation of autophagosomes, expression of heterochromatin marker HP1γ, energy metabolism and cytokine secretion to validate that OSKMLN truly had rejuvenative effects and can reverse cellular aging.

 

Lastly, they performed an in vivo experiment to further support their finding. They isolated young and aged mouse-derived skeletal muscle stem cells and treated them with OSKMLN before transplanting the cells into injured tibialis anterior muscles of immunocompromised mice. Treated skeletal muscle stem cells resulted in improved tissue regenerative potential than aged skeletal muscle stem cells.

 

Overall, the paper showed convincing data that introducing aged cells with a simple cocktail of mRNAs can revert aging phenotypes and can even improve regenerative capacity of cells.

1.     Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease (Bin Ramli et al., 2020; Gastroenterology)

 

This paper describes a relatively simple method to generate liver organoids in a high-throughput method. After human pluripotent stem cells were differentiated into hepatic endoderm spheroids and then further on to hepatoblast spheroids, the hepatoblast spheroids were dissociated and then re-seeded into 96 well plates before continuing their differentiation into hepatic organoids. The authors showed beautiful images of the organoids containing ALB (hepatocytes) and CK7 (cholangiocytes/ bile duct cells). They also deployed RNA-sequencing to further characterize the organoids. What is impressive is that each of the organoids has a well-defined bile caniculi network that is functional and capable of transporting fluorescent compounds.The authors proceeded on to use their organoids to model drug-induced cholestasis (using troglitazone) and fatty liver diseases (using a cocktail of free fatty acids). RNA sequencing data showed that the liver organoids exposed to free fatty acids clustered with the samples obtained from non-alcoholic steatohepatitis (NASH) patients. These organoids also showed clear disruption in the bile caniculi network and loss of polarity amongst hepatocytes. Overall, the authors are convinced that their liver organoids will be able to serve as good cellular models for studying NASH and non-alcoholic fatty liver disease (NAFLD).

 

2.     Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes (Buikema et al., 2020; Cell Stem Cell)

 

This simple and straight-forward paper is from Sean Wu’s group right here in Stanford’s Cardiovascular Institute, School of Medicine. They described a method to expand hiPSC-derived CMs in vitro, a startling 100- to 250-fold expansion, using a combination of GSK-3β inhibition and cell contact removal i.e. passaging cells sparsely. They provided strong evidences indicating that cell-cell contact is a ‘major barrier to hiPSC-CM proliferation’. Using their method, they were able to generate 300-900 million CMs from a starting number of 2 million hiPSC-CM (by the 5th passage) and the purity of these CMs remains to be more than 90% TNT+ by the 4th passage.Next, they moved on to understand the mechanisms underlying this expansion process. They noted that inhibiting GSK-3β in their CMs resulted in a complete sarcomere disassembly, indicative of a reversion to an immature cell state. This is unsurprising because we (Fan et al., 2018) and others have shown that differentiated cells often regress into a ‘dedifferentiated’ state when proliferating. They even provided cool videos showing single-cell contractions.They then performed single cell-RNA sequencing on the proliferative CMs and noted that Wnt target genes such as AXIN2 and LEF1 were upregulated while mature cardiac genes were downregulated. An important result from sequencing data is that the proliferative CMs did not show an upregulation in cardiac progenitor markers indicating that the CMs did not adopt a ‘progenitor state’ during proliferation. Whilst the single cell clusters seems to show a larger population of ventricular CM, the authors did not address much about that.This has profound implications in the field of regenerative therapy because now, we can expand hiPSC-CM on a dish, creating a large pool of cells for tissue engineering and transplantation therapy. The next steps are to transplant these cells (or engineered heart tissues made from these cells) into a mice model of myocardial infarction to see if these cells can engraft and restore cardiac function. It will also be interesting to know if this expansion method will work for human primary CMs and also if the expansion has any ventricular/ atrial-specific preferences.

 

3.     A Multiplex Human Pluripotent Stem Cell Platform Defines Molecular and Functional Subclasses of Autism-Related Genes (Cederquist et al., 2020; Cell Stem Cell)

 

Using cell-based approaches to study autism has always been both challenging and controversial because autism manifests itself primarily as a behavioural deficit, in another words, how to you study disruptions in social-emotional functioning in a dish? Nonetheless, there have been breakthroughs over the past decade in utilizing genome-wide sequence analysis (GWAS) in the hunt for autism-related genes. However, there has yet been studies actively correlating these mutations to the disease phenotypes.This paper describes a unique method to study cellular and molecular abnormalities in neural cells differentiated from patient-derived iPSCs, specifically, cells of the prefrontal cortex (PFC) which is one of the key area of the brain relevant to autism pathology. Using known genetic information associated with autism, the authors constructed 30 disease pluripotent stem cell lines. They then went to pool these disease lines together before  differentiating them to PFC cells.They showed that about 50% of the autism cell lines showed abnormal PFC neurogenesis (i.e. reduced neural output), several cell lines showed neural stem cell enrichment (seemingly indicating a failure to differentiate and mature), several others showed an opposite phenotype. Overall, these results seem to indicate that autism mutations resulted in a dysregulation of proliferation and neural differentiation. The authors highlighted the WNT/ β-catenin signaling pathway is involved in the abnormalities observed i.e. some autism cell lines were hyporesponsive to CHIR-induced stem cell proliferation. At the end of the paper, the authors used the phenotypes PFC development and WNT signaling to cluster autistic patient profiles (2 main clusters) i.e. one group of autistic patients clustered based on abnormal early PFC neurogenesis was associated with increased severity in communication deficits.Altogether, this is a good paper that describes a multiplex platform to study autism-associated mutations, essentially correlating and classifying interesting disease phenotypes to the genotypes. The authors also made use of their results to, in an unbiased manner, group autistic patients into cohorts.

1. Generation of Functional Liver Sinusoidal Endothelial Cells from Human Pluripotent Stem-Cell-Derived Venous Angioblasts (Gage et al., 2020; Cell Stem Cell)

 

Long-anticipated paper from Gordon Keller’s group describing the generation of liver-specific endothelial cells (LSECs) from human pluripotent stem cells (hPSCs). There are several reasons why I am personally excited about this paper: 1. Gordon Keller himself gave a talk about this particular work during the 4th Annual Center for Definitive and Curative Medicine (CDCM) Symposium, organized by Stanford School of Medicine early March this year It is crazy to think that that was shortly before the COVID19 pandemic swept through the country. 2. I worked in a vascular biology lab for a year and continue to work on ECs for another 1.5 years thereafter so any papers on ECs interest me. 3. Because the identities of organ-specific ECs are only recently explored (and remains uncertain), any claim of being able to produce them from hPSCs warrants extra attention.

 

The authors utilized a 3D embryoid body-based protocol wherein cells are patterned towards the mesodermal fate and subsequently to angioblasts, which are supposed precursors of ECs. The angioblasts contains both arterial-like and venous-like cells. The authors then enriched for venous angioblasts, the source of LSECs. The ‘magic’ of said protocol is the manipulation of VEGF-A, bFGF and Notch signaling pathways coupled with cAMP supplementation, TGF-b inhibition and hypoxia.

 

After the differentiation process, they obtained LSECs marked by STAB2 and FCGR2B expression. LSECs engrafted very well in livers of neonatal and adult NSG mice, with engraftment more broadly distributed throughout the adult mice liver. After transplantation, the LSECs also further developed to produce more coagulation factor F8. The authors proceeded on to perform sc-RNA Seq and functionally characterized them (impressive assay to test fenestrations and scavenging potential of LSECs).

 

2.     Hair-bearing human skin generated entirely from pluripotent stem cells (Lee et al., 2020, Nature)

​

I am really excited to share this paper because it literally is like something out of a sci-fi film.  As far as I am concerned, this is the first ever paper describing the generation of human skin that actually presents functional hair follicles from hPSCs. Given that I am a complete novice in any thing ectoderm-related, I thoroughly enjoyed reading this paper.

​

The authors intentionally created skin organoids containing a mix of surface ectodermal cells and mesenchymal cells. After 18 days, the shape of the organoids began to morph and polarize (with a head and tail) and after around 50 days, the epithelium in the organoids became stratified, suggesting complex epithelial organization as with actual skin. They continued differentiation for over 70 days for the organoids to begin growing hair germ-like buds (see cover photo).  After 100 days, the organoids showed compelling similarities to the human fetal skin and showed significant pigmentation. The authors also showed evidence indicating the presence of neural innervation in these organoids. Lastly, the authors transplanted these organoids in small incision sites made at the back skin of nude mice (lacks hair) and slightly more than half of the grafts showed growth of 2-5mm hairs (up to 14 days of observation). Sebaceous glands were also detected in these hair follicles.

​

Whilst the maximal length of hair and longevity of these hair and hair follicles remains untested, this paper is truly ground-breaking and provides a promising treatment option for hair loss in patients especially due to medical/ genetic reasons.

 

3.     Human Cerebrospinal Fluid Promotes Neuronal Circuit Maturation of Human Induced Pluripotent Stem Cell-Derived 3D Neural Aggregates. (Izsak et al., 2020; Stem Cell Reports)

​

Researchers are getting more and more innovative when it comes to promoting maturation of cells in culture; from playing around with different extracellular matrices (ECMs), stimulating cells with drugs and even electrical currents to even exposing cells to cellular stresses. This German group apparently added human cerebrospinal fluid (CSF), that is fluid that flows through our nervous system (sometimes colloquially termed as ‘brain juice’). After generating 3D neural aggregates from iPSCs (using previously publish protocol, which I will not dwell too much on), the authors treated these aggregates with the human CSF.

​

Human CSF samples were obtained from 13 different healthy donors via a procedure known as lumbar puncture. The authors stated that all of the CSF samples caused improvement in neuronal networks all within a short 72h time frame. They are even cultured the aggregates in CSF for almost a month, but noted that the neuronal parameters plateau off at 11 days. CSF treatment also accelerated neuronal differentiation (illustrated by the striking neurite outgrowth) and synapse development (illustrated by dot-like synaptic VGlut sub-cellular localization).  

​

The authors even quantified improvements in synaptic properties and neural connectivity in these aggregates. This paper, however, is mostly descriptive (which probably explains why it is published in Stem Cell Reports), the question of how/why CSF promotes maturation and significantly improves several neural parameters remains unanswered. It will also be interesting to know how these CSF-treated aggregates will integrate/ perform better when transplanted into an animal model. Overall, this paper truly highlights the lab’s strength and specialty in performing neural assays and measuring/ interpreting electrical signals using the complex multi-electrode array (MEA) system.