Linan Liu

A splice donor mutation in NAA10 results in the dysregulation of the retinoic acid signalling pathway and causes Lenz microphthalmia syndrome

Introduction Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition. Methods and results Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells. Conclusions We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway.

From Blood to the Brain: Can Systemically Transplanted Mesenchymal Stem Cells Cross the Blood-Brain Barrier?

Systemically infused mesenchymal stem cells (MSCs) are emerging therapeutics for treating stroke, acute injuries, and inflammatory diseases of the central nervous system (CNS), as well as brain tumors due to their regenerative capacity and ability to secrete trophic, immune modulatory, or other engineered therapeutic factors. It is hypothesized that transplanted MSCs home to and engraft at ischemic and injured sites in the brain in order to exert their therapeutic effects. However, whether MSCs possess the ability to migrate across the blood-brain barrier (BBB) that separates the blood from the brain remains unresolved. This review analyzes recent advances in this area in an attempt to elucidate whether systemically infused MSCs are able to actively transmigrate across the CNS endothelium, particularly under conditions of injury or stroke. Understanding the fate of transplanted MSCs and their CNS trafficking mechanisms will facilitate the development of more effective stem-cell-based therapeutics and drug delivery systems to treat neurological diseases and brain tumors.

Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro.

The delivery of therapeutics to the central nervous system remains a major challenge in part due to the presence of the blood–brain barrier (BBB). Recently, cell-derived vesicles, particularly exosomes, have emerged as an attractive vehicle for targeting drugs to the brain, but whether or how they cross the BBB remains unclear. Here, we investigated the interactions between exosomes and brain microvascular endothelial cells (BMECs) in vitro under conditions that mimic the healthy and inflamed BBB in vivo. Transwell assays revealed that luciferase-carrying exosomes can cross a BMEC monolayer under stroke-like, inflamed conditions (TNF-α activated) but not under normal conditions. Confocal microscopy showed that exosomes are internalized by BMECs through endocytosis, co-localize with endosomes, in effect primarily utilizing the transcellular route of crossing. Together, these results indicate that cell-derived exosomes can cross the BBB model under stroke-like conditions in vitro. This study encourages further development of engineered exosomes as drug delivery vehicles or tracking tools for treating or monitoring neurological diseases.

DNA-Scaffolded Multivalent Ligands to Modulate Cell Function

We report a simple, versatile, multivalent ligand system that is capable of specifically and efficiently modulating cell‐surface receptor clustering and function. The multivalent ligand is made of a polymeric DNA scaffold decorated with biorecognition ligands (i.e., antibodies) to interrogate and modulate cell receptor signaling and function. Using CD20 clustering‐mediated apoptosis in B‐cell cancer cells as a model system, we demonstrated that our multivalent ligand is significantly more effective at inducing apoptosis of target cancer cells than its monovalent counterpart. This multivalent DNA material approach represents a new chemical biology tool to interrogate cell receptor signaling and functions and to potentially manipulate such functions for the development of therapeutics.

Mechanoresponsive stem cells to target cancer metastases through biophysical cues

Targeting the mechano-niche of the tumor microenvironment is a strategy to interrogate, detect, and treat cancer. A stiff punishment for tumors In patients, tumor cells do not grow in isolation, and their behavior is regulated not only by their own biology but also by interactions with their microenvironment. A key part of the microenvironment is the extracellular matrix, which typically has a greater stiffness in tumors than in surrounding normal tissues. To take advantage of this, Liu et al. engineered mechanoresponsive mesenchymal stem cells to act as vehicles for cancer drug delivery. These engineered stem cells accumulated in tumors, delivering the first half of a two-part cancer therapy: the enzyme cytosine deaminase. A drug called 5-fluorocytosine was then delivered systemically, and cytosine deaminase in the tumors activated the drug, providing local anticancer activity with no off-target damage in mice. Despite decades of effort, little progress has been made to improve the treatment of cancer metastases. To leverage the central role of the mechanoenvironment in cancer metastasis, we present a mechanoresponsive cell system (MRCS) to selectively identify and treat cancer metastases by targeting the specific biophysical cues in the tumor niche in vivo. Our MRCS uses mechanosensitive promoter–driven mesenchymal stem cell (MSC)–based vectors, which selectively home to and target cancer metastases in response to specific mechanical cues to deliver therapeutics to effectively kill cancer cells, as demonstrated in a metastatic breast cancer mouse model. Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer–associated mechano-cues. MRCS has markedly reduced deleterious effects compared to MSCs constitutively expressing therapeutics. MRCS indicates that biophysical cues, specifically matrix stiffness, are appealing targets for cancer treatment due to their long persistence in the body (measured in years), making them refractory to the development of resistance to treatment. Our MRCS can serve as a platform for future diagnostics and therapies targeting aberrant tissue stiffness in conditions such as cancer and fibrotic diseases, and it should help to elucidate mechanobiology and reveal what cells “feel” in the microenvironment in vivo.

Exogenous marker-engineered mesenchymal stem cells detect cancer and metastases in a simple blood assay

IntroductionMesenchymal stem cells (MSCs) are adult multipotent stem cells that possess regenerative and immunomodulatory properties. They have been widely investigated as therapeutic agents for a variety of disease conditions, including tissue repair, inflammation, autoimmunity, and organ transplantation. Importantly, systemically infused MSCs selectively home to primary and metastatic tumors, though the molecular mechanisms of tumor tropism of MSCs remain incompletely understood. We have exploited the active and selective MSCs homing to cancer microenvironments to develop a rapid and selective blood test for the presence of cancer.MethodsWe tested the concept of using transplanted MSCs as the basis for a simple cancer blood test. MSCs were engineered to express humanized Gaussia luciferase (hGluc). In a minimally invasive fashion, hGluc secreted by MSCs into circulation as a reporter for cancer presence, was assayed to probe whether MSCs co-localize with and persist in cancerous tissue.ResultsIn vitro, hGluc secreted by engineered MSCs was detected stably over a period of days in the presence of serum. In vivo imaging showed that MSCs homed to breast cancer lung metastases and persisted longer in tumor-bearing mice than in tumor-free mice (P < 0.05). hGluc activity in blood of tumor-bearing mice was significantly higher than in their tumor-free counterparts (P < 0.05).ConclusionsBoth in vitro and in vivo data show that MSCs expressing hGluc can identify and report small tumors or metastases in a simple blood test format. Our novel and simple stem cell-based blood test can potentially be used to screen, detect, and monitor cancer and metastasis at early stages and during treatment.

A mathematical model of mechanotransduction reveals how mechanical memory regulates mesenchymal stem cell fate decisions

BackgroundMechanical and biophysical properties of the cellular microenvironment regulate cell fate decisions. Mesenchymal stem cell (MSC) fate is influenced by past mechanical dosing (memory), but the mechanisms underlying this process have not yet been well defined. We have yet to understand how memory affects specific cell fate decisions, such as the differentiation of MSCs into neurons, adipocytes, myocytes, and osteoblasts.ResultsWe study a minimal gene regulatory network permissive of multi-lineage MSC differentiation into four cell fates. We present a continuous model that is able to describe the cell fate transitions that occur during differentiation, and analyze its dynamics with tools from multistability, bifurcation, and cell fate landscape analysis, and via stochastic simulation. Whereas experimentally, memory has only been observed during osteogenic differentiation, this model predicts that memory regions can exist for each of the four MSC-derived cell lineages. We can predict the substrate stiffness ranges over which memory drives differentiation; these are directly testable in an experimental setting. Furthermore, we quantitatively predict how substrate stiffness and culture duration co-regulate the fate of a stem cell, and we find that the feedbacks from the differentiating MSC onto its substrate are critical to preserve mechanical memory. Strikingly, we show that re-seeding MSCs onto a sufficiently soft substrate increases the number of cell fates accessible.ConclusionsControl of MSC differentiation is crucial for the success of much-lauded regenerative therapies based on MSCs. We have predicted new memory regions that will directly impact this control, and have quantified the size of the memory region for osteoblasts, as well as the co-regulatory effects on cell fates of substrate stiffness and culture duration. Taken together, these results can be used to develop novel strategies to better control the fates of MSCs in vitro and following transplantation.

Facile supermolecular aptamer inhibitors of L-selectin.

Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.

Targeting Biophysical Cues: a Niche Approach to Study, Diagnose, and Treat Cancer

Probing the biophysical properties of the tumor niche offers a new perspective in cancer mechanobiology, and supports the development of next-generation diagnostics and therapeutics for cancer, in particular for metastasis.

Enhanced Therapeutic Effects of Mesenchymal Stem Cell-Derived Exosomes with an Injectable Hydrogel for Hindlimb Ischemia Treatment

Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidates for cell-free treatment of various diseases. However, maintaining the retention and stability of exosomes over time in vivo after transplantation is a major challenge in the clinical application of MSC-derived exosomes. Here, we investigated if human placenta-derived MSC-derived exosomes incorporated with chitosan hydrogel could boost the retention and stability of exosomes and further enhance their therapeutic effects. Our results demonstrated that chitosan hydrogel notably increased the stability of proteins and microRNAs in exosomes, as well as augmented the retention of exosomes in vivo as confirmed by Gaussia luciferase imaging. In addition, we assessed endothelium-protective and proangiogenesis abilities of hydrogel-incorporated exosomes in vitro. Meanwhile, we evaluated the therapeutic function of hydrogel-incorporated exosomes in a murine model of hindlimb ischemia. Our data demonstrated that chitosan hydrogel could enhance the retention and stability of exosomes and further augment the therapeutic effects for hindlimb ischemia as revealed by firefly luciferase imaging of angiogenesis. The strategy used in this study may facilitate the development of easy and effective approaches for assessing and enhancing the therapeutic effects of stem cell-derived exosomes.