Develop a 3D neurological disease model of human cortical glutamatergic neurons using micropillar-based scaffolds

Establishing an effective three-dimensional (3D) culture system to better model human neurological diseases is desirable, since the human brain is a 3D structure. Here, we demonstrated the development of a polydimethylsiloxane (PDMS) pillar-based 3D scaffold that mimicked the 3D microenvironment of the brain. We utilized this scaffold for the growth of human cortical glutamatergic neurons that were differentiated from human pluripotent stem cells. In comparison with the 2D culture, we demonstrated that the developed 3D culture promoted the maturation of human cortical glutamatergic neurons by showing significantly more MAP2 and less Ki67 expression. Based on this 3D culture system, we further developed an disease-like model of traumatic brain injury (TBI), which showed a robust increase of glutamate-release from the neurons, in response to mechanical impacts, recapitulating the critical pathology of TBI. The increased glutamate-release from our 3D culture model was attenuated by the treatment of neural protective drugs, memantine or nimodipine. The established 3D human neural culture system and TBI-like model may be used to facilitate mechanistic studies and drug screening for neurotrauma or other neurological diseases.

Single-cell qPCR facilitates the optimization of hematopoietic differentiation in hPSCs/OP9 coculture system

Human pluripotent stem cells (hPSCs)/OP9 coculture system is a widely used hematopoietic differentiation approach. The limited understanding of this process leads to its low efficiency. Thus, we used single-cell qPCR to reveal the gene expression profiles of individual CD34+ cells from different stages of differentiation. According to the dynamic gene expression of hematopoietic transcription factors, we overexpressed specific hematopoietic transcription factors (Gata2, Lmo2, Etv2, ERG, and SCL) at an early stage of hematopoietic differentiation. After overexpression, we generated more CD34+ cells with normal expression level of CD43 and CD31, which are used to define various hematopoietic progenitors. Furthermore, these CD34+ cells possessed normal differentiation potency in colony-forming unit assays and normal gene expression profiles. In this study, we demonstrated that single-cell qPCR can provide guidance for optimization of hematopoietic differentiation and transient overexpression of selected hematopoietic transcription factors can enhance hematopoietic differentiation.

New Advances in Human X Chromosome Status from a Developmental and Stem Cell Biology

Recent advances in stem cell biology have dramatically increased the understanding of molecular and cellular mechanism of pluripotency and cell fate determination. Additionally, pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells, arose as essential resources for disease modeling and cellular therapeutics. Despite these advancements, the epigenetic dysregulation in pluripotency such as the imprinting status, and X chromosome dosage compensation, and its consequences on future utility of PSCs yet remain unresolved. In this review, we will focus on the X chromosome regulation in human PSCs (hPSCs). We will introduce the previous findings in the dosage compensation process on mouse model, and make comparison with those of human systems. Particularly, the X chromosome activation status of human preimplantation embryos, and the regulation of the active X chromosome by human specific lincRNA, X Active Coating Transcript (XACT), will be discussed. We will also discuss the recent findings on higher order X chromosome architecture, and abnormal X chromosome status in hPSCs.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Engineering Biomaterials for Feeder-Free Maintenance of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) are capable of differentiating into any type of somatic cell, a characteristic that imparts significant therapeutic potential. Human embryonic stem cells and induced pluripotent stem cells are types of hPSCs. Although hPSCs have high therapeutic potential, their clinical relevance is limited by the requirement for animal feeder layers, which maintain their pluripotency and self-renewal. hPSCs grown on animal feeder cells are at high risk for pathogen contamination and can be affected by the immunogenicity of the feeder layer. The presence of animal feeder cells also limits the scalability of hPSCs in culture because of the high cost of culturing and batch-to-batch variations. Therefore, development of feeder-free systems is imperative for robust, lower-cost, xeno-free, scalable culture of hPSCs. Biomaterials engineered with bioactive molecules such as adhesion proteins and extracellular matrix proteins, or synthetic materials such as peptides and polymers, may provide alternative substrates to animal feeder cells. This article reviews biomaterial-based, feeder-free systems for hPSC growth and maintenance, which provide clinically relevant alternatives to feeder cell systems.