Jiang F. Zhong

Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter

We report a high speed and high purity pulsed laser triggered fluorescence activated cell sorter (PLACS) with a sorting throughput up to 20,000 mammalian cells/s with 37% sorting purity, 90% cell viability in enrichment mode, and >;90% purity in high purity mode at 1,500 cells/s or 3,000 beads/s. Fast switching (30 μs) and a small perturbation volume (~90 pL) is realized by a unique sorting mechanism in which explosive vapor bubbles are generated using focused laser pulses in a single layer microfluidic PDMS channel.

Highly Parallel Genome-Wide Expression Analysis of Single Mammalian Cells

Background We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs. Methodology/Principal Findings The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R2∼0.76–0.80 between individual cells and R2∼0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R2∼0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input. Conclusions/Significance In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology.

Increase developmental plasticity of human keratinocytes with gene suppression

Recent evidence indicates that p53 suppression increased the efficiency of induced pluripotent stem cell (iPSC) generation. This occurred even with the enforced expression of as few as two canonical transcription factors, Oct4 and Sox2. In this study, primary human keratinocytes were successfully induced into a stage of plasticity by transient inactivation of p53, without enforced expression of any of the transcription factors previously used in iPSC generation. These cells were later redifferentiated into neural lineages. The gene suppression plastic cells were morphologically indistinguishable from human ES cells. Gene suppression plastic cells were alkaline phosphatase-positive, had normal karyotypes, and expressed p53. Together with the accumulating evidence of similarities and overlapping mechanisms between iPSC generation and cancer formation, this finding sheds light on the emerging picture of p53 sitting at the crossroads between two intricate cellular potentials: stem cell vs. cancer cell generation. This finding further supports the crucial role played by p53 in cellular reprogramming and suggests an alternative method to switch the lineage identity of human cells. This reported method offers the potential for directed lineage switching with the goal of generating autologous cell populations for novel clinical applications for neurodegenerative diseases.

Microfluidic Devices for High-Throughput Gene Expression Profiling of Single hESC-Derived Neural Stem Cells

Isolating pure stem cell populations is one of the major obstacles in stem cell gene expression profiling due to the lack of stem cell markers. Many results of gene expression profiling studies are difficult to interpret because of the heterogeneous cell populations used in these studies. Single-cell gene expression profiling is perhaps the most attractive gene expression profiling method for studying stem cell gene regulation, because isolating pure stem cell population is not needed. However, current single-cell gene expression profiling methods such as laser capture microdissection (LCM) and patch-clamp analysis lack the high-throughput ability in sample processing. For better understanding of the gene regulation networks during cellular events, a large number of gene expression profiles are required. Therefore, we developed inexpensive microfluidic devices for high-throughput single-cell gene expression profiling. With our devices, cDNA could be obtained from 50 individual cells within 3 hours. This approach can be applied to neural stem cells, and other cell types.

Microfluidic Devices for Investigating Stem Cell Gene Regulation via Single-Cell Analysis

The underlying gene interactions that collectively govern the regulation of cellular events can be studied by simultaneous measurement of the levels of mRNA from multiple involved genes. Ideally a single cell should be studied, for comparison with other cells, like and unlike. However, conventional gene expression profiling techniques require several thousands or even millions of cells in order to obtain a sufficient amount of mRNA for analysis. Obtaining this number of cells of a single type is difficult, especially in attempting to study rare stem cells. Single-cell gene expression profiling can, in theory, overcome this limitation. However, conventional analytic methods and equipment are not suitable for analyzing a single-cell, which has a volume of only one or two picoliters. Inexpensive microfluidic devices that can precisely manipulate several nanoliters of fluids are, in theory, ideal for single-cell analysis. By performing biochemical reactions in small volumes, microfluidic devices also minimize material loss in single-cell analysis. This review describes current microfluidic technology applied to single-cell analysis, with a primary focus upon study of single mammalian stem cells. Microfluidic devices have the potential to transform single-cell analysis from a major technological challenge task to a relatively routine procedure for research and clinical assays.

Microfluidic Polymerase Chain Reaction

We implement microfluidic technology to miniaturize a thermal cycling system for amplifying DNA fragments. By using a microfluidic thermal heat exchanger to cool a Peltier junction, we have demonstrated rapid heating and cooling of small volumes of solution. We use a miniature K-type thermocouple to provide a means for in situ sensing of the temperature inside the microrefrigeration system. By combining the thermocouple, two power supplies controlled by a relay system, and computer automation, we reproduce the function of a commercial polymerase chain reaction thermal cycler and demonstrate amplification of a DNA sample of about 1000 base pairs.

A real-time pluripotency reporter for human stem cells.

Pluripotency of stem cells refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm. Maintaining pluripotent stem cells in culture is a tedious and demanding task. Monitoring the changing pluripotency in live cells is essential for this task. Here, we report a pluripotency monitoring system in which the expression of green fluorescent protein (GFP) is under the control of the promoter of a pluripotency gene (Rex-1). The reporter system can be permanently integrated into the genome of live cells via lentiviral vectors. This pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single cell and its progeny. Our data demonstrate that the BJ cell line (a normal human fibroblast cell line) that carries this hRex-GFP construct does not express GFP until it is reprogrammed to pluripotent stage. The GFP expression was progressively lost when these pluripotent hRex-GFP cells exposed to differentiation conditions. These results indicate that insertion of the hRex-GFP construct is stable in descendant cells, a finding that has particular value in tracking pluripotency of transplanted cells and their progenies in animal studies. With this hRex-GFP reporter, the pluripotency of cells can be monitored over long periods of time via the expression of GFP. Use of this reporter system will facilitate the study of stem cell pluripotency at the single-cell level, and sheds light on the molecular mechanisms of stem cell self-renewal and subsequent differentiation.

Epigenomic Reprogramming of Adult Cardiomyocyte-Derived Cardiac Progenitor Cells

It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration.

Increased expression of CX43 on stromal cells promotes leukemia apoptosis

Connexin 43 (Cx43) induced apoptosis has been reported in solid tumors, but the effect of Cx43 expressed by bone marrow stromal cells (BMSC) in leukemia has not been fully investigated. Manipulating Cx43 expression could be a potential therapeutic strategy for leukemia. Here, we investigate the effect of Cx43 expressed by BMSCs (human Umbilical Cord Stem Cells over-expressed CX43, Cx43-hUCSC) on leukemia cells. When co-cultured with Cx43-hUCSC, leukemia cells show significant lower growth rate with increasing apoptosis activity, and more leukemia cells enter S phase. Functional assays of fluorescence recovery after photo bleaching (FRAP) showed improved gap junctional intercellular communication (GJIC) on leukemia cells when co-cultured with Cx43-hUCSC (p < 0.01). In a mouse minimal disease model, the mean survival time and mortality rate were significantly improved in mice transplanted with Cx43-hUCSC. Our results indicate that Cx43 expressed by BMSC induces apoptosis on leukemia cells. Small molecules or other pharmaceutical approaches for modulating Cx43 expression in BMSCs could be used for delaying relapse of leukemia.

Human-derived normal mesenchymal stem/stromal cells in anticancer therapies

The tumor microenvironment (TME) not only plays a pivotal role during cancer progression and metastasis, but also has profound effects on therapeutic efficacy. Stromal cells of the TME are increasingly becoming a key consideration in the development of active anticancer therapeutics. However, dispute concerning the role of stromal cells to fight cancer continues because the use of mesenchymal stem/stromal cells (MSCs) as an anticancer agent is dependent on the specific MSCs subtype, in vitro or in vivo conditions, factors secreted by MSCs, types of cancer cell lines and interactions between MSCs, cancer cells and host immune cells. In this review, we mainly focus on the role of human-derived normal MSCs in anticancer therapies. We first discuss the use of different MSCs in the therapies for various cancers. We then focus on their anticancer mechanism and clinical application.