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-induced differentiation in hematopoietic stem cells.

Aging of hematopoietic stem cells (HSCs) is accompanied by diminished functional potential. Wang et al. now provide evidence for an HSC-specific differentiation checkpoint mediated by the transcription factor BATF, which limits self-renewal of HSCs in response to the accumulation of DNA damage.

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.

The H2 S Dimer is Hydrogen-Bonded: Direct Confirmation from Microwave Spectroscopy.

Ice and solid H2 S look as different as pears and oranges, leading Pauling to conclude that H2 O has hydrogen bonds and H2 S has van der Waals interactions. Now it is shown that the H2 S dimer, like the H2 O dimer, is indeed hydrogen-bonded.

Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-ß receptor tyrosine kinase signaling.

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1(-/-)) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-ß receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-ß receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.

Mitochondrial thioredoxin reductase is essential for early postischemic myocardial protection.

BACKGROUND: Excessive formation of reactive oxygen species contributes to tissue injury and functional deterioration after myocardial ischemia/reperfusion. Especially, mitochondrial reactive oxygen species are capable of opening the mitochondrial permeability transition pore, a harmful event in cardiac ischemia/reperfusion. Thioredoxins are key players in the cardiac defense against oxidative stress. Mutations in the mitochondrial thioredoxin reductase (thioredoxin reductase-2, Txnrd2) gene have been recently identified to cause dilated cardiomyopathy in patients. Here, we investigated whether mitochondrial thioredoxin reductase is protective against myocardial ischemia/reperfusion injury. METHODS AND RESULTS: In mice, α-MHC-restricted Cre-mediated Txnrd2 deficiency, induced by tamoxifen (Txnrd2-/-ic), aggravated systolic dysfunction and cardiomyocyte cell death after ischemia (90 minutes) and reperfusion (24 hours). Txnrd2-/-ic was accompanied by a loss of mitochondrial integrity and function, which was resolved on pretreatment with the reactive oxygen species scavenger N-acetylcysteine and the mitochondrial permeability transition pore blocker cyclosporin A. Likewise, Txnrd2 deletion in embryonic endothelial precursor cells and embryonic stem cell-derived cardiomyocytes, as well as introduction of Txnrd2-shRNA into adult HL-1 cardiomyocytes, increased cell death on hypoxia and reoxygenation, unless N-acetylcysteine was coadministered. CONCLUSIONS: We report that Txnrd2 exerts a crucial function during postischemic reperfusion via thiol regeneration. The efficacy of cyclosporin A in cardiac Txnrd2 deficiency may indicate a role for Txnrd2 in reducing mitochondrial reactive oxygen species, thereby preventing opening of the mitochondrial permeability transition pore.

Murine HSCs contribute actively to native hematopoiesis but with reduced differentiation capacity upon aging.

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age.

A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.

Recent evidence points to the embryonic emergence of some tissue-resident innate immune cells, such as B-1a lymphocytes, prior to and independently of hematopoietic stem cells (HSCs). However, whether the full hematopoietic repertoire of embryonic HSCs initially includes these unique lineages of innate immune cells has been difficult to assess due to lack of clonal assays that identify and assess HSC precursor (pre-HSC) potential. Here, by combining index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays, we analyze emerging pre-HSCs at the single-cell level, revealing their unique stage-specific properties and clonal lineage potential. Remarkably, clonal pre-HSCs detected between E9.5 and E11.5 contribute to the complete B cell repertoire, including B-1a lymphocytes, revealing a previously unappreciated common precursor for all B cell lineages at the pre-HSC stage and a second embryonic origin for B-1a lymphocytes.

Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing.

Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.

Genome editing for human gene therapy.

The rapid advancement of genome-editing techniques holds much promise for the field of human gene therapy. From bacteria to model organisms and human cells, genome editing tools such as zinc-finger nucleases (ZNFs), TALENs, and CRISPR/Cas9 have been successfully used to manipulate the respective genomes with unprecedented precision. With regard to human gene therapy, it is of great interest to test the feasibility of genome editing in primary human hematopoietic cells that could potentially be used to treat a variety of human genetic disorders such as hemoglobinopathies, primary immunodeficiencies, and cancer. In this chapter, we explore the use of the CRISPR/Cas9 system for the efficient ablation of genes in two clinically relevant primary human cell types, CD4+ T cells and CD34+ hematopoietic stem and progenitor cells. By using two guide RNAs directed at a single locus, we achieve highly efficient and predictable deletions that ablate gene function. The use of a Cas9-2A-GFP fusion protein allows FACS-based enrichment of the transfected cells. The ease of designing, constructing, and testing guide RNAs makes this dual guide strategy an attractive approach for the efficient deletion of clinically relevant genes in primary human hematopoietic stem and effector cells and enables the use of CRISPR/Cas9 for gene therapy.

Fgd5 identifies hematopoietic stem cells in the murine bone marrow.

Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence.

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 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.

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 Parkinson's 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.

Expenditure to treat thalassaemia: an experience at a tertiary care hospital in India.

BACKGROUND: The medical and economic problem of thalassaemia are considered to be a vast public health problem in the thalassaemia belt countries, emphasizing more on prenatal diagnosis as the solution of the problem. METHODS: A cross-sectional descriptive study was conducted in the Institute of Haematology & Transfusion Medicine located in Medical College, Kolkata, India to assess the socio-demographic profile, clinical presentation, expenditure for treatment of thalassaemia patients and awareness about cause and prevention of the disease. RESULTS: Thalassaemia patients attended the Govt. setting were mostly from lower socioeconomic status with low level of literacy. Annual expenditure for treatment of thalassaemia ranged from $ 108 to 432; depending on type of treatment with average cost per transfusion was $ 5.2±2.2. Average 18.5%±14.3 of the total annual income was spent on the treatment for thalassaemia. Average man days or school days lost for the patients was 29.87±18.5 and 19.07±12.7 for the accompanying persons. CONCLUSION: Blood transfusion and carrier screening facilities should be decentralized to decrease the expenditure for treatment and alleviate the harassment of the families. Folate and calcium tablets, hepatitis B vaccination can be made available at government setting free of cost.

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.

Plasmon-phonon coupling in charged n-type CdSe quantum dots: A THz time-domain spectroscopic study.

This work aims to experimentally determine the polarizability of confined electron in CdSe quantum dots (QD). The dielectric response of uncharged and charged CdSe quantum dots (3.2 and 6.3 nm) has been measured using terahertz time-domain spectroscopy in the frequency range of 2.0-7.0 THz. A strong coupling between the surface plasmon and surface phonons appears upon charging the QDs. The absolute polarizability of an electron in 3.2 and 6.3 nm charged QDs are experimentally determined to be 0.5 +/- 0.1 x 10(3) A3 and 14.6 +/- 0.3 x 10(3) A3, respectively, and the values agree reasonably well with theory and the previous experiment. The observed plasmon-phonon coupling is expected to play an important role in electron relaxation in absence of a hole in CdSe QDs.

Ab initio and AIM theoretical analysis of hydrogen-bond radius of HD (D = F, Cl, Br, CN, HO, HS and CCH) donors and some acceptors.

Recently, we defined 'hydrogen-bond radii' for various hydrogen-bond donors, DH where D = F, Cl, Br, CN, HO or CCH from an empirical analysis. It was shown that the A...H distances in B...HD complexes could be written as a sum of hydrogen bond radius for DH and a constant acceptor radius for A, which is the bonding atom/centre in B. This manuscript reports the determination of the hydrogen-bond radii for these molecules and H2S from ab initio and atoms in molecules (AIM) theoretical calculations. The results from ab initio calculations are consistent with the empirical estimates for the six molecules noted above and provide the first estimate for hydrogen bond radius (1.08 +/- 0.16 A) for H2S donor. The results from AIM theoretical analysis are in qualitative agreement with ab initio results. However, AIM analysis indicates that both hydrogen bond donor and acceptor radii vary in a systematic way from the strong to weak hydrogen bonds. Irrespective of the method used, the hydrogen bond donor radius increases in the order HF < HCl < H2O < HBr < HCN < HCCH < H2S, but mostly lie between Pauling's covalent and van der Waals radii of H atom. Interestingly, the acceptor radii for A in A...HD also increase in the same order. The AIM theoretical results on about 100 complexes have been reduced to suggest radii for H, F, O, N, C and S that are appropriate for strong, medium and weak hydrogen bonds. It is suggested that the use of a single van der Waals radius for D, H or A in determining the presence/absence of D-H...A hydrogen bonding be discontinued.

Rotational spectra and structure of the Ar2-H2S complex: pulsed nozzle Fourier transform microwave spectroscopic and ab initio studies.

This paper reports the rotational spectrum and structure of the Ar2-H2S complex and its HDS and D2S isotopomers. The ground state structure has heavy-atom C2v symmetry with the two Ar atoms indistinguishable and H2S freely rotating as evinced by the fact that asymmetric top energy levels with Kp=odd levels are missing. The rotational constants for the parent isotopomer are: A=1733.115(1) MHz, B=1617.6160(5) MHz and C=830.2951(2) MHz. Unlike the Ar-H2S complex, the Ar2-H2S does not show an anomalous isotopic shift in rotational constants on deuterium substitution. However, the intermolecular potential is still quite floppy, leading to very different centrifugal distortion constants for the three isotopomers. The Ar-Ar and Ar-c.m.(H2S) distances are determined to be 3.820 A and 4.105 A, respectively. The A rotational constants for Ar2-H2S/HDS/D2S isotopomers are very close to each other and to the B constant of free Ar2, indicating that H2S does not contribute to the moment of inertia about the a-axis. Ab initio calculations at MP2 level with aug-cc-pVQZ basis set lead to an equilibrium C2v minimum structure with the Ar-Ar line perpendicular to the H-H line and the S away from Ar2. The centrifugal distortion constants, calculated using the ab initio force field, are in reasonable agreement with the experimental values. However, they do not show the variation observed for different isotopmers. The binding energy of Ar2-H2S has been determined to be 507 cm-1(6.0 kJ mol-1) by CBS extrapolation after correcting for basis set superposition error. Potential energy scans point out that the barrier for internal rotation of H2S about its b axis is only 10 cm-1 and it is below the zero point energy (13.5 cm-1) in this torsional degree of freedom. Internal rotation of H2S about its a- and c-axes also have small barriers of about 50 cm-1 only, suggesting that H2S is extremely floppy within the complex.