Pankaj K. Mandal

Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine.

Human iPSC-Based Modeling of Late-Onset Disease via Progerin-Induced Aging

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinsons disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.

Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.

Reprogramming Committed Murine Blood Cells to Induced Hematopoietic Stem Cells with Defined Factors

Hematopoietic stem cells (HSCs) sustain blood formation throughout life and are the functional units of bone marrow transplantation. We show that transient expression of six transcription factors Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37 imparts multilineage transplantation potential onto otherwise committed lymphoid and myeloid progenitors and myeloid effector cells. Inclusion of Mycn and Meis1 and use of polycistronic viruses increase reprogramming efficacy. The reprogrammed cells, designated induced-HSCs (iHSCs), possess clonal multilineage differentiation potential, reconstitute stem/progenitor compartments, and are serially transplantable. Single-cell analysis revealed that iHSCs derived under optimal conditions exhibit a gene expression profile that is highly similar to endogenous HSCs. These findings demonstrate that expression of a set of defined factors is sufficient to activate the gene networks governing HSC functional identity in committed blood cells. Our results raise the prospect that blood cell reprogramming may be a strategy for derivation of transplantable stem cells for clinical application.

DNA damage response in adult stem cells: pathways and consequences

In contrast to postmitotic or short-lived somatic cells, tissue-specific stem cells must persist and function throughout life to ensure tissue homeostasis and repair. The enormous functional demands and longevity of stem cells raises the possibility that stem cells might be uniquely equipped to maintain genomic integrity in ways different than somatic cells. Indeed, evidence suggests that stem cell compartments possess unique properties that combine to either limit or, in some instances, accelerate DNA damage accrual.

Reprogramming human fibroblasts to pluripotency using modified mRNA

Induced pluripotent stem (iPS) cells hold the potential to revolutionize regenerative medicine through their capacity to generate cells of diverse lineages for future patient-specific cell-based therapies. To facilitate the transition of iPS cells to clinical practice, a variety of technologies have been developed for transgene-free pluripotency reprogramming. We recently reported efficient iPS cell generation from human fibroblasts using synthetic modified mRNAs. Here we describe a stepwise protocol for the generation of modified mRNA–derived iPS cells from primary human fibroblasts, focusing on the critical parameters including medium choice, quality control, and optimization steps needed for synthesizing modified mRNAs encoding reprogramming factors and introducing these into cells over the course of 2–3 weeks to ensure successful reprogramming. The protocol described herein is for reprogramming of human fibroblasts to pluripotency; however, the properties of modified mRNA make it a powerful platform for protein expression, which has broad applicability in directed differentiation, cell fate specification and therapeutic applications.

System xc− and Thioredoxin Reductase 1 Cooperatively Rescue Glutathione Deficiency

GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in γ-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation.

Loss of Thioredoxin Reductase 1 Renders Tumors Highly Susceptible to Pharmacologic Glutathione Deprivation

Tumor cells generate substantial amounts of reactive oxygen species (ROS), engendering the need to maintain high levels of antioxidants such as thioredoxin (Trx)- and glutathione (GSH)-dependent enzymes. Exacerbating oxidative stress by specifically inhibiting these types of ROS-scavenging enzymes has emerged as a promising chemotherapeutic strategy to kill tumor cells. However, potential redundancies among the various antioxidant systems may constrain this simple approach. Trx1 and thioredoxin reductase 1 (Txnrd1) are upregulated in numerous cancers, and Txnrd1 has been reported to be indispensable for tumorigenesis. However, we report here that genetic ablation of Txnrd1 has no apparent effect on tumor cell behavior based on similar proliferative, clonogenic, and tumorigenic potential. This finding reflects widespread redundancies between the Trx- and GSH-dependent systems based on evidence of a bypass to Txnrd1 deficiency by compensatory upregulation of GSH-metabolizing enzymes. Because the survival and growth of Txnrd1-deficient tumors were strictly dependent on a functional GSH system, Txnrd1-/- tumors were highly susceptible to experimental GSH depletion in vitro and in vivo. Thus, our findings establish for the first time that a concomitant inhibition of the two major antioxidant systems is highly effective in killing tumor, highlighting a promising strategy to combat cancer.

Fgd5 identifies hematopoietic stem cells in the murine bone marrow

Fdg5 identifies bone marrow cells with potent hematopoietic stem cell activity.

Terahertz spectroscopy studies on epitaxial vanadium dioxide thin films across the metal-insulator transition

We present results on terahertz (THz) spectroscopy on epitaxial vanadium dioxide (VO(2)) films grown on sapphire across the metal-insulator transition. X-ray diffraction indicates the VO(2) film is highly oriented with the crystallographic relationship: (002)(film)//(0006)(sub) and [010](film)//[2 ̅1 ̅10](sub). THz studies measuring the change in transmission as a function of temperature demonstrate an 85% reduction in transmission as the thin film completes its phase transition to the conducting phase, which is much greater than the previous observation on polycrystalline films. This indicates the crucial role of microstructure and phase homogeneity in influencing THz properties.