Abstract
Multiscale Microenvironmental Perturbation of Pluripotent Stem Cell Fate and Self-Organization
Stem cell behavior is strongly influenced by extrinsic factors from the microenvironmental niche, yet it is highly challenging to elucidate their roles. Because stem cell fate is triggered by highly complex spatial and temporal displays of microenvironmental signals, cell culture platforms that exert close control over microenvironmental conditions are necessary. Existing microfluidic systems are poorly suited for long-term stem cell maintenance and subsequent organoid development. Other factors, such as shear stresses, depleted autocrine factors, medium evaporation, and overly specialized equipment systems, further hamper widespread usage. The authors of this report present an easy-to-use microchip system to control biomolecular spatiotemporal profiles. These can be tethered to hydrogels and presented in a graded manner or integrated with “feeder” cell–supporting compartments to study long-range cell–cell communication.
As a proof of concept, mouse embryonic stem cells (mESCs) are cultured in three-dimensional (3D) conditions and induced toward the neural lineage while exposed to a leukemia inhibitory factor (LIF, a self-renewal factor) present in soluble, cell-secreted, or gel-immobilized form. The biomicrofluidic chip comprises either one or two hydrogel compartments (depending on whether the feeder cell compartment is necessary). The presentation and mass transfer of protein growth factors can also be controlled by cross-linked hydrogels. This generates growth factor gradients that can be adjusted in diffusion speed and steepness, which is confirmed by computer simulations.
To recapitulate the manner in which regulatory proteins are present in extracellular matrix (ECM) biology, LIF molecules are Fc tagged (from immunoglobulin moieties) and immobilized on 3D poly(ethylene glycol) (PEG)–based hydrogels. With such tools, graded displays of LIF protein factors are generated to examine mESC choice of whether to self-renew or be induced into neural differentiation.
Rex1::GFP mESCs (which emit a signal when the pluripotent factor is expressed) are compared between engineered LIF gradients to factors supplied by feeder cells. Whereas Rex1 expression levels decrease without supplied LIF factors, both engineered and feeder-supplied LIF conditions lead to mESCs with graded green fluorescent protein (GFP) expression, with the greatest intensity located near its source. In contrast, under gel-immobilized LIF conditions, the pluripotency status is lost within a 4-day culture period.
Taken together, a novel microengineered platform for 3D stem cell culture is demonstrated that can control the presentation of growth factors critical to stem cell identity in three ways: soluble molecules, cell-secreted molecules, and ECM-immobilized molecules. The results bring about a novel hypothesis arising from experimental observations, such as the nature of the LIF (growth factor), cell-borne LIF receptor interactions, and LIF consumption. By controlling the presentation of critical regulatory factors (such as LIF), these platforms exert more exquisite control over the distribution, the fate of stem cells, their microenvironment, and cell/fate patterning. (Tabata, Y.; Lutolf, M. P. Sci. Rep.
Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells
Even though astrocytes are the most numerous cell lineage in the mammalian brain, little is known about them. They are known to play many critical roles, such as synapse formation and neurotransmitter recycling. Astrocytes are also known to exist in at least two distinct states (i.e., fetal and mature) with thousands of different genes expressing differences. These developmental changes occur throughout gestation until early postnatal development and are potentially critical for neuropsychiatric disease development and the role of glia in brain disorders. Investigating astrocyte maturation in primary brain tissue is challenging and sample availability is sparse, especially during critical brain developmental periods.
Induced pluripotent stem cell (iPSC) cultures allow the generation of astrocytes in two-dimensional (2D) cultures, albeit limited to shorter culture periods. In response, the authors of this report utilize 3D cultures that maintain spheroid cultures for 20 months and beyond. Critically, these cultures can recapitulate key features of in vivo cortical development. Cryosections of the cortical spheroids show glial fibrillary acidic protein (GFAP) reminiscent of astrocytes. To isolate astrocyte lineage cells, spheroid tissue is dissociated into single cells and plated onto cell culture plates coated with antibodies for CD90 (neuronal cell capture) and glial cell adhesion molecule (HepaCAM—astrocyte lineage capture) antibodies. Cultures of these CD90 and HepaCAM isolated cells lead to neuron and astrocyte lineages, respectively, following longer culture periods.
Principal component analysis (PCA) using established transcriptomic datasets further demonstrates that the isolated astrocyte-like cells cluster closely to fetal and mature primary astrocytes. Exploiting the long-term potential of spheroid culture, the authors profile the gene expression of HepaCAM isolated cells between days 100 and 590. Genes such as AQP4, ALDH1L1, and RANBP3L (“mature” astrocyte genes) increase in expression levels, whereas TOP2A and TMSB15A (“fetal” astrocyte genes) decrease accordingly. This and the microarray data suggest that long-term culture leads to the fetal transcriptomic program deactivating and a progressive transformation into mature astrocytes.
The authors proceed to probe the HepaCAM isolated astrocytes using a series of functional assays. Radioactively labeled glutamate is found to be efficiently taken up. Mouse brain-derived synaptosomes are also readily phagocytosed. These also induce synapse formation when cultured with retinal ganglion cells and facilitated neuronal cell maturation. Given the ability for long-term brain tissue–like spheroid culture, this resource can mature astrocytic cells into varying stages of development. Critically, these are functional, and potentially model various neurodevelopmental conditions, such as autism spectrum disorders and schizophrenia. (Sloan, S. A.; et al. Neuron
CryoPause: A New Method to Immediately Initiate Experiments after Cryopreservation of Pluripotent Stem Cells
Pluripotent stem cells (PSCs) are well known for their potential in disease modeling and cell replacement therapy; however, numerous aspects regarding their maintenance and differentiation workflow can still be improved. Often, synchronizing PSC culture is challenging because they expand and differentiate at different rates. During continuous passage, several issues potentially occur, such as spontaneous differentiation, risk of contamination with other cells/microorganisms, likely selection for highly proliferative cell mutants, and an increased likelihood of human error.
Unlike current protocols that continuously expand PSCs in colonies and necessitate the usage of later passage colonies for differentiation into requisite therapeutic lineages, the authors of this report propose the CryoPause (CP) method, which expands PSCs into a large pool over the least number of passages. This batch is dissociated into ready-to-use single-cell suspension aliquots before cryopreservation, which permits the initiation of PSC experiments using the same starting material.
WA09 human embryonic stem cells (hESCs) are expanded in Essential 8 medium before Accutase treatment to create a single-cell suspension suspended in FreSR-S (a commercial cryopreservative) and frozen in liquid nitrogen. Postthaw cells remain highly viable with no observable change for up to 1 year of cryostorage.
To validate CP PSCs, select pluripotency and spontaneous differentiation markers are measured (flow cytometry of immunomarkers) and found to be similar to control PSCs. Whole-genome transcriptional profiles (PluriTest) are also within the safe threshold scores. Bisulfide sequencing also reveals that the epigenetic status is similar between CP and control PSCs. Using functional teratoma assays, CP PSCs have similar differentiation properties to control PSCs, forming derivatives of all three germ layers. Postthaw DNA damage is an issue previously reported. γH2AX immunofluorescence (DNA damage biomarker) finds no statistical difference between CP and control PSC expression. The CP PSCs are not more susceptible to DNA damage following exposure to cytotoxic alkaloids. The CP protocol also does not generate karyotypic abnormalities.
To validate differentiation performance, CP PSCs are also readily converted to progenitor and mature neural lineages like control PSCs. In addition, the scale-up (0.25 billion cells per factory) and genetic modification potential (Sendai viral vectors, CRISPR and HPRT-deleted mutants) is also validated. The critical contribution of the CP protocol is to eliminate potential variability in starting material quality (seeded PSCs). This CP protocol supports off-the-shelf PSC bioprocessing by assisting the provision of patient human leukocyte antigen (HLA)–matched PSC-based therapeutics. (Wong, K. G.; et al. Stem Cell Rep.
A Portable Paper-Based Microfluidic Platform for Multiplexed Electrochemical Detection of Human Immunodeficiency Virus and Hepatitis C Virus Antibodies in Serum
Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) are among the leading causes of global morbidity and mortality. Furthermore, coinfection exists to a significant extent in HIV patients and further weakens them. Although point-of-care (POC) tests for sexually transmitted infections (STIs) are commercially available, they are often not affordable or accessible to patients in the developing world. Existing technology is typically designed to diagnose a binary status (positive or negative) and cannot quantify disease marker concentration to determine infection stage.
In this report, Zhao and Liu propose an electrochemical microfluidic paper analytical device. Exploiting electron transfer from redox reactions provides higher sensitivity, a lower limit of detection, and diverse sensing capabilities for POC testing. This platform integrates a paper-based sensor array with a handheld potentiostat to perform enzyme-linked immunosorbent assay (ELISA) on eight serum samples in parallel, generating results in 20 min and transmitting these via computing or mobile communication devices. Crucially, diagnosis of HIV/HCV coinfection is feasible due to the negligible cross-reactivity between the assays.
For specific HIV and HCV detection, an indirect ELISA working principle is exploited. HIV or HCV capture antigens are tethered to the device reaction zone before sample incubation for 3 min. Thereafter, alkaline phosphatase–labeled secondary antibodies are bound to the captured primary HIV/HCV antibodies before p-aminophenyl phosphate (pAPP) is added, which generates an amperometric current output. The entire process allows eight samples to be read within 20 min for prepared devices.
In spiked mouse serum samples, the limit of detection for HIV and HCV antibodies is determined to be 0.3 and 0.75 ng/mL compared with existing tests (1 and 5 ng/mL, respectively). This electrochemical microfluidic approach successfully measures concentrations of antibody over six orders of magnitude. Critically, samples spiked with high concentrations of interfering antibodies (1000-fold higher) do not reduce its diagnostic fidelity. Further work is required to improve the long-term stability of this device (potentially by adding protein stabilizers), to trial this platform on patient samples, and apply it to other diagnostic targets. (Zhao, C.; Liu, X. Biomicrofluidics
Circulating ECV-Associated miRNAs as Potential Clinical Biomarkers in Early-Stage HBV- and HCV-Induced Liver Fibrosis
Liver fibrosis is the resultant pathology of sustained wound healing due to chronic liver injury (including hepatitis B/C viral infections). This process is associated with activation of hepatic stellate cells (HSCs), which leads to excessive deposition and accumulation of liver ECM. Liver fibrosis progresses to cirrhosis (loss of organ function) and hepatocellular carcinoma (HCC). Liver biopsies are the gold standard, but they are invasive and associated with pain, bleeding, and other severe complications. While other noninvasive techniques have been utilized, they do not identify the early stages of disease progression or discriminate between the various stages.
Analysis of circulating microRNAs (miRNAs) has emerged as a less invasive diagnostic method. Studies support this notion, as a higher incidence of circulating vesicles have been observed in human patients and mouse disease models. Lambrecht et al. propose that HSC activation results in a circulating miRNA signature that reflects the stage of liver fibrosis resulting from HBV/HCV infections.
A cohort of 39 patients with HBV and HCV infections were enrolled and compared with 14 healthy individuals without liver disease symptoms or history. All patients underwent fibroscan (transient elastography) to exclude patients with advanced stage liver disease. Based on established information, miRNAs 150, 192, and 200b were selected as potential liver fibrosis biomarkers, with miRNA 122 chosen as a positive control. miRNAs 92a and 21 are chosen for their known presence in pathological conditions and potential implication in liver disease.
Among the various miRNA species, miRNA 122 is higher in patient groups (validating previous reports). miRNA 192 is higher in HBV but not HCV patients, and miRNA 200b is higher in both early-stage HBV and HCV patients. Among miRNAs 192, 150, 92a, and 200b, receiver operating characteristic (ROC) plots identify all the species that have a strong discriminative potential between healthy and fibrotic HBV and HCV patients (area under curve, mostly >0.95).
To associate miRNA secretion patterns, vesicles from primary culture–activated mouse HSCs are collected, and miRNA content also validates the diagnostic value of the miRNA panel. While the patient cohort is considered relatively small, the panel of miRNAs suggests they are suitable as a diagnostic panel for early-stage liver fibrosis. Further work using unbiased RNA sequencing could identify other potential disease biomarkers. Circulating miRNAs may even provide updates regarding the status of fibrosis resolution and gauge the effect of antifibrotic drugs. (Lambrecht, J.; et al. Front. Pharmacol.
A Versatile Microfluidic Device for High-Throughput Production of Microparticles and Cell Microencapsulation
Biocompatible microparticles are involved in numerous biomedical applications, such as controlled drug release, analytical assays, and cell-based therapy. Although typical processes involve the cross-linking of a monomer solution and gentle handling of live cellular organisms, the shear rate of current industrial systems is too high, at least 100-fold higher than the shear rate in blood capillaries. Microfluidics is a feasible technique to create such cell droplets, but current systems are considered insufficiently reliable (jetting and clogging issues). Efforts to increase the system throughput include parallelizing droplet generation in the order of 100 s. However, issues of droplet stability, cross talk, and reproducibility still plague these designs.
Akbari and colleagues describe a design that continuously generates microdroplets by breaking an aqueous stream over an array of micropillars at a high frequency of 3.1 MHz (three orders of magnitude higher than single-flow focusing). Droplet diameter can be tuned between 8 and 65 µm by adjusting flow rates and tuning droplet physicochemical properties. The first few rows of micropillars serve to focus flow into larger droplets, before the subsequent droplets are homogenized over the remainder of micropillars until they reach a critical size that stably passes through the pillars without further decomposition into smaller droplets. This platform facilitates the generation of various microdroplet material chemistries, such as poly(ethylene glycol) diacrylate (PEGDA), calcium alginate, and chemically cross-linked PEG microparticles.
As a proof of concept, MDA-MB231 breast cancer cells are encapsulated in PEG gels functionalized with GRGDSP peptides. Although initial viability of the encapsulated cells decreases to 68% (using a calcein AM live-cell assay), this proportion increases to 80% after 13 days due to cell proliferation. This system reliably generates microparticles containing single cells. Importantly, this is a highly robust and cost-effective microfluidic platform that can generate up to 75 g of microparticles per day, amenable to various photopolymerization, ionic, and chemical cross-linking mechanisms. (Akbari, S.; et al. Lab Chip
Membrane Pore Spacing Can Modulate Endothelial Cell–Substrate and Cell–Cell Interactions
As interest in mimicking native cell behavior in vitro grows, cell culture systems such as organ-on-chip, membrane-supported barrier models, and coculture systems have grown in popularity. These potentially translate into reduced drug development costs and may replace animal models with sophisticated human cell systems. Understanding various cell–cell and cell–substrate interactions is thus critical to improved development of human cell/organ-on-chip systems. Cell–substrate interactions are mediated by mechanical forces transmitted through cell anchoring known as focal adhesions (FAs). FAs can vary to a large extent with respect to the stiffness of the substrate (stiffer substrates have longer FA, whereas softer substrates have shorter FA).
By using a combination of different culture substrates, the effect of membrane pore spacing on cell–substrate and corresponding cell–cell interactions is investigated in this report. Using ultrathin (300 nm thick) glass SiO2 membranes, the authors can visualize cellular FAs and quantify the number of FAs. The SiO2 membranes are fabricated to have pores that prevent (0.5 µm) or allow (3.0 µm) cellular migration. These are also performed alongside other control substrates, such as tissue culture plastic, as well as stiff and soft polydimethylsiloxane (PDMS) substrates.
The authors next investigate the formation of endothelial barriers on their system of culture substrates. A greater density of pores with smaller diameter (0.5 µm) reduces the quantity of FA fibrils, which are even lower than those of the soft PDMS controls. Having established that higher pore density (0.5 µm) results in weaker cell–substrate interactions (evidenced by lower FA expression and shorter fibrils), they investigate the relationship with cell–cell interactions.
At 96 h of culture, the tight junctional protein (representative of cell–cell interactions) ZO-1 is assayed. Whereas the stiffer and nonporous substrates express weaker expression levels, soft PDMS and the 0.5 µm pore (greater pore density) substrates exhibit robust ZO-1 expression. This is consistent with the notion that weak cell–substrate interactions lead to strong cell–cell interactions. Measuring the alignment of cellular fibers further confirms this view, as the 0.5 µm pore substrates display more oriented alignment than the nonporous and larger (less dense) pore spacing.
Using fabricated substrates with well-controlled culture parameters sheds further light regarding cell–substrate and cell–cell interactions. These provide further understanding and enable better-informed design parameters for miniaturized human cell culture systems. (Casillo, S. M.; et al. Biomater. Sci. Eng.
Topical Tissue Nanotransfection Mediates Nonviral Stroma Reprogramming and Rescue
In vivo cell reprogramming has the potential to be a highly transformative therapy. Existing methods of reprogramming heavily rely on viral transfection that are not amenable to clinical translation. Viral capsid size and the stochastic nature of reprogramming further limit their efficacy. Herein, the authors report a novel nonviral approach to topically reprogram tissues in situ using a nanochanneled device—tissue nanotransfection (TNT). TNT uses a highly intense and focused electric field that directly transfers reprogramming DNA plasmid factors into cell cytosol. TNT is also topically administered for a short period of time (<1 s), using multiple pulses of 10 ms to deliver the factors. In contrast to bulk electroporation that generates inflammation and cell death, TNT allows focused delivery of greater quantities of plasmid factors safely.
As a proof of concept, overexpressing the factors ADCL/BRN2/MYT1L (ABM) induces fibroblasts to become neuronal (induced neurons [iNs]). Interestingly, TNT demonstrates fate conversion beyond the physical transfection boundary. This is found to be a result of cell-released extracellular vehicles (EVs) rich with cDNA/mRNA factors. As further evidence of EV-generated TNT reprogramming, partially converted iNs are found to express COL1 and keratin 14, suggesting that iNs are of dermal and epidermal origin, respectively. Within live mice, iNs can be traced to skin cells, with ectopic Tuj1 expression found within the hair follicular regions. Converted iNs are characterized by transcriptome profiling, and tested for neuronal excitability (electrophysiology).
Having validated the transdermal plasmid factor delivery capabilities in converting neurons, the authors next use this platform for reprogramming skin to induced endothelial cells. This is achieved using the factors ETV2, FOXC2, and FLI1 (EFF). Following in vitro validation, TNT delivery of EFF is found to increase the expression of PECAM-1 and von Willebrand factor (vWF) in dorsal skin stroma, suggesting increased endothelial lineage conversion. High-resolution laser speckle and ultrasound imaging shows that TNT-based EFF delivery enhances blood flow within 3 days, even showing that the newly formed vessels merge with the arteries (anastomosis). To examine the therapeutic effects of TNT delivery of endothelial lineage factors, they show increased blood flow in ischemic tissue flaps that prevent tissue necrosis.
This is further extended to whole-limb rescue experiments that involve transecting the femoral artery. Within 7 days of TNT, perfusion is shown to be higher, less tissue necrosis is observed in treated limbs, and muscle energetics (increased ATP and phosphocreatine levels) improve. Due to the effect of EVs, angiogenesis is also induced far beyond the treatment location. Further analysis confirms their presence and suggests their role in niche preconditioning by spreading pro-angiogenic signals even within the initial posttransfection period. An intriguing possibility suggests that the TNT method can be used to generate autologous cells within the patient’s own tissue and harvested for cell replacement therapy. (Gallego-Perez, D.; et al. Nat. Nanotechnol.
FolamiRs: Ligand-Targeted, Vehicle-Free Delivery of miRNAs for the Treatment of Cancer
Although miRNAs are very ideal as therapeutic candidates, their practical translation into efficient therapeutics is still limited. Issues such as delivery-associated toxicity, low transfection efficiency, systemic clearance, nonspecific biodistribution, and degradation within circulation are hindrances to achieving effective therapeutics. While vehicle-free delivery methods have been proposed for miRNA mimics, they are not amenable to stabilizing modifications that prevent intracellular loading and recognition. Common miRNA therapeutics involve packaging into nanoparticle, liposomal, micelle, and other biomaterial delivery vehicles.
To overcome such challenges, cell surface receptors can be exploited for targeted and focused miRNA delivery. The folate receptor (FR) is an attractive candidate because it is overexpressed on many cancer cells relative to healthy cells. It also has a suitable ligand—vitamin B9 (folic acid), which suggests that FR conjugate therapy has great potential for small RNA (miRNA and siRNA) delivery.
To create a miRNA–conjugate therapeutic, a miR-34a strand (miR-34a-5p) is conjugated to folate-dibenzocyclooctyne (Fol-DBCO) using either unreleasable or releasable conformations (FolamiRs). Cellular data show that both folate conjugates (tagged with fluorescence dyes) are efficiently taken up by FR-positive cell lines (MB-231—human breast adenocarcinoma–derived cells), whereas this is not observed for FR-negative ones (A549). Further assays using a miR-34a Renilla luciferase sensor confirm functional downregulation in MB-231 cells. Further studies show that both releasable and unreleasable FolamiRs efficiently enter cells, retaining activity without interfering with miR-34a-5p intracellular interference mechanisms (Argonaute processing).
To assess the in vivo performance of FolamiRs, these are injected into mice bearing MB-231 sensor cell xenografts. At 24 h postinjection, NIR-FolamiR is mostly retained in tumor tissues but cleared from the rest of the organism. However, functional activity (measured by Renilla activity in vivo) is limited to the unreleasable NIR-FolamiR-34a. Releasable FolamiRs are presumed to have been degraded or prematurely reduced in circulation.
Furthermore, the specificity of the miR conjugate strategy in vivo is demonstrated by blocking its accumulation on MB-231 xenografted mice using excess folate-glucosamine. In contrast, A549 xenograft mice do not show the same accumulation. Over a 20-day dosing period, the miR-34a FolamiR conjugates reduce MB-231 xenograft size by ~40%. Crucially, there is no evidence of whole-organ toxicity or elevation in serum interleukin-6 and tumor necrosis factor-α.
Thereafter, the FolamiR strategy is applied to a Kras/p53 non-small-cell lung cancer model (NSCLC), which is a relevant, orthotopic mouse cancer model with intact immune system. It not only accurately recapitulates NSCLC progression but also develops resistance to conventional therapy. In this model, FolamiR-34a conjugates similarly suppressed tumor growth compared with noncoding controls and reduced the extent of lung tumor burden. Direct measurement of miR-34a in excised lung tumor tissue shows that it is threefold higher than that in control samples. Target genes such as BCL-2 and MYC are also suppressed, confirming miR-34a activity.
This approach demonstrates that conjugates targeting ligands with miR species facilitate efficient translation. miRNA therapy maximizes high uptake in cancer cells, has minimal side effects, and simultaneously targets multiple pathways due to their pleiotropic nature, which reduces acquired resistance. Conjugated miRNAs also remove vehicle-associated toxicity. However, by exploiting cell receptor uptake, future generations need to incorporate endosomal escape mechanisms to increase delivery payload. (Orellana, E. A.; et al. Sci. Transl. Med.