Background & Aims-Liver transplantation is the primary treatment for various end-stage hepatic diseases but is hindered by the lack of donor organs and by complications associated with rejection and immunosuppression. There is increasing evidence to suggest the bone marrow is a transplantable source of hepatic progenitors. We previously reported that multipotent bone marrow-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells with almost 100% induction frequency under defined conditions, suggesting the potential for clinical applications. The aim of this study was to critically analyze the various parameters governing the success of bone marrow-derived mesenchymal stem cell-based therapy for treatment of liver diseases.

We utilized a system for sequence-specific RNA base editing via Adenosine Deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). We systematically engineered it to harness ADARs, and comprehensively evaluated its specificity and activity in vitro and in vivo via two mouse models of human disease. We anticipate this platform will enable tunable and reversible engineering of RNAs for diverse applications.

We reconstituted type I collagen nanofibers prepared by electrospin technology and examined the morphology, growth, adhesion, cell motility, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (

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Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner.

To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this paper we present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. One salient feature of the assay is the non-invasive collection and surveying of single cell secretions at different time points, producing unprecedented insight of single cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

The effect of substrate-mediated signals on osteogenic differentiation of hMSCs is studied using a synthetic bone-like material comprising both organic and inorganic components that supports adhesion, spreading, and proliferation of hMSCs. hMSCs undergo osteogenic differentiation even in the absence of osteogenesis-inducing supplements. They exhibit higher expressions of Runx2, BSP, and OCN compared to their matrix-rigidity-matched, non-mineralized hydrogel counterparts. The mineralized-hydrogel-assisted osteogenic differentiation of hMSCs could be attributed to their exposure to high local concentrations of calcium and phosphate ions in conjunction with chemical and topological cues arising from the hydrogel-bound calcium phosphate mineral layer.

Cell-responsive hydrogels hold tremendous potential as cell delivery devices in regenerative medicine. In this study, we developed a hydrogel-based cell delivery vehicle, in which the encapsulated cell cargo control its own release from the vehicle in a protease-independent manner. Specifically, we have synthesized a modified poly(ethylene glycol) (PEG) hydrogel that undergoes degradation responding to cell-secreted molecules by incorporating disulfide moieties onto the backbone of the hydrogel precursor. Our results show the disulfide-modified PEG hydrogels disintegrate seamlessly into solution in presence of cells without any external stimuli. The rate of hydrogel degradation, which ranges from hours to months, is found to be dependent upon the type of encapsulated cells, cell number, and fraction of disulfide moieties present in the hydrogel backbone. The differentiation potential of human mesenchymal stem cells released from the hydrogels is maintained in vitro. The in vivo analysis of these cell-laden hydrogels, through a dorsal window chamber and intramuscular implantation, demonstrated autonomous release of cells to the host environment. The hydrogel-mediated implantation of cells resulted in higher cell retention within the host tissue when compared to that without a biomaterial support. Biomaterials that function as a shield to protect cell cargos and assist their delivery in response to signals from the encapsulated cells could have a wide utility in cell transplantation and could improve the therapeutic outcomes of cell-based therapies.