A microfluidic culture for two populations of dorsal root ganglia for differential staining

The goal of this study was to design and fabricate a microfluidic system that can be used to visibly distinguish the two populations of dorsal root ganglia (DRGs) by differential staining. Polydimethylsiloxane (PDMS) is the most widely used silicon-based organic polymer, and is particularly known for its wide spread use in microfluidics. Various methods have been employed to pump fluids in these channels for applications ranging from patterning of cells and biomolecules to control of local environment factors such as temperature, which requires external pumping or other applied forces. We demonstrated a pump-free device that exploits the surface energy stored in a liquid droplet to pump liquid in the channels. The fluid was pumped by using two droplets of unequal sizes connected via fluid filled channel. The flow was generated from smaller droplet to larger droplet. This passive pumping technique was used to simultaneously stain the two cultured DRGs in connected channels. The in vitro system can be further exploited to study the guided growth in axons. This study provides a cost effective method to detect the influence of the presence of pioneer neuron on the growth patterns of the new generation of neurons. It eliminates the need of using transgenic cells to study the guided growth in axons, thereby giving some insight for the repair of spinal cord injuries and the understanding of the early growth model

Harnessing the Power of Induced Pluripotent Stem Cells and Gene Editing Technology: Therapeutic Implications in Hematological Malignancies

In vitro modeling of hematological malignancies not only provides insights into the influence of genetic aberrations on cellular and molecular mechanisms involved in disease progression but also aids development and evaluation of therapeutic agents. Owing to their self-renewal and differentiation capacity, induced pluripotent stem cells (iPSCs) have emerged as a potential source of short in supply disease-specific human cells of the hematopoietic lineage. Patient-derived iPSCs can recapitulate the disease severity and spectrum of prognosis dictated by the genetic variation among patients and can be used for drug screening and studying clonal evolution. However, this approach lacks the ability to model the early phases of the disease leading to cancer. The advent of genetic editing technology has promoted the generation of precise isogenic iPSC disease models to address questions regarding the underlying genetic mechanism of disease initiation and progression. In this review, we discuss the use of iPSC disease modeling in hematological diseases, where there is lack of patient sample availability and/or difficulty of engraftment to generate animal models. Furthermore, we describe the power of combining iPSC and precise gene editing to elucidate the underlying mechanism of initiation and progression of various hematological malignancies. Finally, we discuss the power of iPSC disease modeling in developing and testing novel therapies in a high throughput setting

Modeling Down Syndrome Myeloid Leukemia by Sequential Introduction of <i>GATA1</i> and <i>STAG2</i> Mutations in Induced Pluripotent Stem Cells with Trisomy 21

Children with Down syndrome (DS) have a high risk for acute myeloid leukemia (DS-ML). Genomic characterization of DS-ML blasts showed the presence of unique mutations in GATA1, an essential hematopoietic transcription factor, leading to the production of a truncated from of GATA1 (GATA1s). GATA1s, together with trisomy 21, is sufficient to develop a pre-leukemic condition called transient abnormal myelopoiesis (TAM). Approximately 30% of these cases progress into DS-ML by acquisition of additional somatic mutations in a stepwise manner. We previously developed a model for TAM by introducing disease-specific GATA1 mutation in trisomy 21-induced pluripotent stem cells (iPSCs), leading to the production of N-terminally truncated short form of GATA1 (GATA1s). In this model, we used CRISPR/Cas9 to introduce a co-operating mutation in STAG2, a member of the cohesin complex recurrently mutated in DS-ML but not in TAM. Hematopoietic differentiation of GATA1 STAG2 double-mutant iPSC lines confirmed GATA1s expression and the loss of functional STAG2 protein, leading to enhanced production of immature megakaryocytic population compared to GATA1 mutant alone. Megakaryocyte-specific lineage expansion of the double-mutant HSPCs exhibited close resemblance to the DS-ML immunophenotype. Transcriptome analysis showed that GATA1 mutation resulted in downregulation of megakaryocytic and erythrocytic differentiation pathways and interferon α/β signaling, along with an upregulation of pathways promoting myeloid differentiation such as toll-like receptor cascade. The co-occurrence of STAG2 knockout partially reverted the expression of genes involved in myeloid differentiation, likely leading to enhanced self-renewal and promoting leukemogenesis. In conclusion, we developed a DS-ML model via hematopoietic differentiation of gene-targeted iPSCs bearing trisomy 21