Chemically Defined Conditions Mediate an Efficient Induction of Dental Pulp Pluripotent-Like Stem Cells into Hepatocyte-Like Cells

Liver diseases are major causes of morbidity and mortality. Dental pulp pluripotent-like stem cells (DPPSCs) are of a considerable promise in tissue engineering and regenerative medicine as a new source of tissue-specific cells; therefore, this study is aimed at demonstrating their ability to generate functional hepatocyte-like cells in vitro. Cells were differentiated on a collagen scaffold in serum-free media supplemented with growth factors and cytokines to recapitulate liver development. At day 5, the differentiated DPPSC cells expressed the endodermal markers FOXA1 and FOXA2. Then, the cells were derived into the hepatic lineage generating hepatocyte-like cells. In addition to the associated morphological changes, the cells expressed the hepatic genes HNF6 and AFP. The terminally differentiated hepatocyte-like cells expressed the liver functional proteins albumin and CYP3A4. In this study, we report an efficient serum-free protocol to differentiate DPPSCs into functional hepatocyte-like cells. Our approach promotes the use of DPPSCs as a new source of adult stem cells for prospective use in liver regenerative medicine.

First person – Viju Vijayan Pillai and Prasanthi Koganti

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Viju Vijayan Pillai and Prasanthi Koganti are co-first authors on ‘ Efficient induction and sustenance of pluripotent stem cells from bovine somatic cells’, published in BIO. Viju conducted the research described within this article while a PhD student in the lab of Vimal Selvaraj at Cornell University, Ithaca, NY, USA, and is now a resident veterinary anatomic pathologist at Purdue University, Indianapolis, IN, USA, investigating reproductive pathology and early embryonic development. Prasanthi is a postdoctoral associate in the lab of Vimal Selvaraj at Cornell University, investigating molecular mechanisms that regulate cellular functions.

Serum but not mucosal antibody responses are predicted by pre-existing SARS-CoV-2 spike cross-reactive CD4+ T cells following BNT162b2 vaccination in the elderly

Advanced age is a main risk factor for severe COVID-19 and thus elderly were often prioritized for vaccination. However, low vaccination efficacy and accelerated waning immunity have been reported in this age group. To elucidate age-related differences in immunogenicity, we analysed cellular, serological and salivary SARS-CoV-2 spike glycoprotein-specific immune responses to BNT162b2 COVID-19 vaccine in old (69-92 years) and middle-aged (24-57 years) vaccinees compared to natural infection (COVID-19 convalescents of 21-55 years). Serological humoral responses to vaccination exceeded those of convalescents but salivary anti-spike subunit 1 (S1) IgA and neutralizing capacity were less durable in vaccinees. In old vaccinees, we observed that pre-existing spike-specific CD4+ T cells correlated with efficient induction of serological anti-S1 IgG and neutralizing capacity after vaccination. Our results highlight the role of pre-existing cross-reactive CD4+ T cells with respect to SARS-CoV-2 vaccination particularly in old individuals, in whom their presence predicted efficient COVID-19-vaccine-induced humoral immune responses.

Targeted biallelic integration of an inducible Caspase 9 suicide gene for safer iPSC-based cellular therapies

The teratoma forming potential of pluripotent stem cells (PSCs), and genetic aberrations that may lead to tumor formation from PSC derivatives, are considered as a major safety risk for cellular therapies. Introduction of inducible suicide genes as synthetic fail-safe systems has been proposed to minimize these risks. Recent research challenged the usefulness of such systems even for targeted introduction via accurate gene editing approaches. Apparently transgene silencing and elimination of a HTK suicide gene through 'loss-of-heterozygosity' (LoH) led to cell clones that escaped the induced suicide. We have introduced an inducible Caspase9 (iCasp-9) suicide gene into induced pluripotent stem cells (iPSCs), that has already been applied clinically in other settings. The iCASP9 gene coupled to a red nuclear GFP variant under control of the CAG promoter was inserted into the AAVS1 locus, either monoallelic or homozygous on both alleles. Efficient induction of apoptosis in vitro could be induced via treatment of iCASP9 iPSCs with two chemical inducers of dimerization (CID) at different concentrations for 24 hours. While NODSCID mice after transplantation of undifferentiated monoallelic iCASP9 iPSCs under the kidney capsule developed teratomas, CID treatment for three days led to rapid shrinking of such tumor structures. In individual mice, however, that received monoallelic iCASP9 iPSCs, tumor-like human tissue could be detected after CID treatment. Further in vitro experiments confirmed that in very rare subclones monoallelic iCASP9 hiPSCs lost transgene expression and can became resistant to CID induction in vitro with frequencies of ~ 3x10^-8. Analysis of CID-resistant subclones identified either elimination of the transgene, presumably via LoH, or via methylation of the CAG promoter as underlying mechanism. In contrast, we never observed any CID resistant escapees form biallelic iCASP9 iPSC clones, even after treatment of up to 0,5x10^9 iPSCs. This observation further argues for LoH as underlying mechanism of transgene elimination in monoallelic clones and suggests that CAG promoter methylation on both alleles represent independent events. In conclusion, biallelic integration of an iCASP9 safety switch in the AAVS1 locus allows for efficient induction of cellular suicide and may substantially increase the safety level of iPSC-based therapies. We propose that safety levels should be calculated by relating the observed frequencies of clonal escapees to clinically relevant cell numbers, i.e. cell number in tumors of a size that is readily detectable by modern imaging approaches.

The thymic microenvironment gradually modulates the phenotype of thymus-homing peripheral conventional dendritic cells

Thymic conventional dendritic cells (t-DCs) are crucial for the development of T cells. A substantial fraction of t-DCs originates extrathymically and migrates to the thymus. Here, these cells contribute to key processes of central tolerance like the clonal deletion of self-reactive thymocytes and the generation of regulatory T (Treg) cells. So far, it is only incompletely understood which impact the thymic microenvironment has on thymus-homing conventional DCs (cDCs), which phenotypic changes occur after the entry of peripheral cDCs into the thymus and which functional properties these modulated cells acquire. In the present study, we mimicked the thymus-homing of peripheral cDCs by introducing ex vivo isolated splenic cDCs (sp-DCs) into re-aggregated thymic organ cultures (RTOCs). Already after two days of culture, the transcriptomic profile of sp-DCs was modulated and had acquired certain key signatures of t-DCs. The regulated genes included immunomodulatory cytokines and chemokines as well as co-stimulatory molecules. After four days of culture, sp-DCs appeared to have at least partially acquired the peculiar Treg cell-inducing capacity characteristic of t-DCs. Taken together, our findings indicate that peripheral cDCs possess a high degree of plasticity enabling them to quickly adapt to the thymus-specific microenvironment. We further provide indirect evidence that thymus-specific properties such as the efficient induction of Treg cells under homeostatic conditions can be partially transferred to thymus-homing peripheral cDC subsets.

Comparison of Analytical and Software Based Design of Energy Efficient Induction Motor

Energy Efficient Induction motor is playing a vital role in current scenario. Due to rising electrical energy demand, increased awareness of global warming, and rising fossil fuel prices, energy efficiency has become increasingly important. Apart from adding capacity, the only practical approach to deal with this situation is to make optimal use of the available energy, which may be done by using energy efficient Motors. The main objective of this paper is to calculate and Compare the Analytical and Electromagnetic Software based design for a 5HP Energy Efficient Motor.

General characteristics of adjuvants and their mechanisms of action (part 2)

One of the major public health challenges today is development of new vaccines and technologies to optimize the vaccination process. There is a growing scientific interest in vaccine adjuvants that enhance vaccine immunogenicity. At present, numerous studies are underway to develop COVID-19 vaccines, including inactivated and subunit vaccines which contain adjuvants for efficient induction of immune response and solid immunity. The aim of the study was to systematise literature related to the analysis of the structure, mechanisms of action and stimulating properties of vaccine adjuvants (synthetic oligodeoxynucleotides, virosomes, polyoxidonium, sovidone), as well as to summarise data on the effects of adjuvants used in SARS-CoV, MERS-CoV, and SARS-CoV-2 vaccine development studies. The paper analyses the prospects for enhancing the stimulating effect of the adjuvants when used in combination with compounds having a different mechanism of action. It also analyses the results of studies of adjuvanted vaccines against SARS-CoV and MERS-CoV, which may be useful when selecting adjuvants with optimal efficacy and safety profiles to be used in SARS-CoV-2 vaccines under development. It was concluded that understanding of the mechanisms of action of adjuvants that mediate their stimulating effect on the body’s immune system will contribute to safe and effective use of adjuvants to enhance the immunogenicity of both authorised and new vaccines.