A method of identifying microorganisms with reduced adaptability to a particular environment, wherein (1) each microorganism is independent by insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence. Mutating to provide a plurality of microorganisms, or clones of said microorganisms, such that each mutation has a different label sequence; (2) providing storage samples of each mutation produced by step (1), respectively, and providing storage nucleic acids each comprising a unique label sequence from each mutation; (3) introducing a plurality of mutations produced by step (1) into the specific environment such that the microorganisms capable of growing in the specific environment grow in the above environment; (4) recovering the microorganisms or selected portions thereof from the environment and separating nucleic acids from the recovered microorganisms; (5) comparing all label sequences in the nucleic acid isolated in step (4) with the unique label sequences of each of the stored mutations as in step (2); and (6) any label isolated in step (4). Screening for individual mutations that also do not have a sequence.

Identification of genes

Human induced pluripotent stem cells (hiPSCs) are an important cell source for regenerative medicine products. Effective methods of preservation are critical to their clinical and commercial applications. The use of a dimethyl sulfoxide (DMSO)-free solution containing all non-toxic molecules offers an effective alternative to the conventional DMSO and alleviates pain points associated with the use of DMSO in the cryopreservation of hiPSCs. Both hiPSCs and cells differentiated from them are commonly multicellular systems, which are more sensitive to stresses of freezing and thawing than single cells. In this investigation, low-temperature Raman spectroscopy visualized freezing behaviors of hiPSC aggregates in different solutions. These aggregates exhibited sensitivity to undercooling in DMSO-containing solutions. We demonstrated the ability to replace DMSO with non-toxic molecules, improve post-thaw cell survival, and reduce sensitivity to undercooling. An accelerated optimization process capitalized on the positive synergy among multiple DMSO-free molecules, which acted in concert to influence ice formation and protect cells during freezing and thawing. A differential evolution algorithm was used to optimize the multi-variable, DMSO-free preservation protocol in 8 experiments. hiPSC aggregates frozen in the optimized solution did not exhibit the same sensitivity to undercooling as those frozen in non-optimized solutions or DMSO, indicating superior adaptability of the optimized solution to different freezing modalities and unplanned deviations. This investigation shows the importance of optimization, explains the mechanisms and advantages of a DMSO-free solution, and enables not only improved cryopreservation of hiPSCs but potentially other cell types for translational regenerative medicine.

/pdf/cryopreservation-of-human-ips-cell-aggregates-in-a-dmso-free-2746xdtfwe.pdf

Cryopreservation of Human iPS Cell Aggregates in a DMSO-Free Solution-An Optimization and Comparative Study.

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.

/pdf/screening-out-irrelevant-cell-based-models-of-disease-4zrtp8alzd.pdf

Screening out irrelevant cell-based models of disease

Matrigel, a basement-membrane matrix extracted from Engelbreth–Holm–Swarm mouse sarcomas, has been used for more than four decades for a myriad of cell-culture applications. However, Matrigel is limited in its applicability to cellular biology, therapeutic-cell manufacturing and drug discovery, owing to its complex, ill-defined and variable composition. Variations in the mechanical and biochemical properties within a single batch of Matrigel — and between batches — have led to uncertainty in cell-culture experiments and a lack of reproducibility. Moreover, Matrigel is not conducive to physical or biochemical manipulation, making it difficult to fine-tune the matrix to promote intended cell behaviours and achieve specific biological outcomes. Recent advances in synthetic scaffolds have led to the development of xenogenic-free, chemically defined, highly tunable and reproducible alternatives. In this Review, we assess the applications of Matrigel in cell culture, regenerative medicine and organoid assembly, detailing the limitations of Matrigel and highlighting synthetic-scaffold alternatives that have shown equivalent or superior results. Additionally, we discuss the hurdles that are limiting a full transition from Matrigel to synthetic scaffolds and provide a brief perspective on the future directions of synthetic scaffolds for cell-culture applications. Matrigel is widely used for cell culture. However, its ill-defined composition, batch-to-batch variability and animal-derived nature lead to experimental uncertainty and a lack of reproducibility. In this Review, we discuss the limitations of Matrigel and highlight synthetic alternatives for stem-cell culture, regenerative medicine and organoid assembly.

Synthetic alternatives to Matrigel.

Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells - a review.

Recent progresses in the field of Induced Pluripotent Stem Cells (iPSCs) have opened up many gateways for the research in therapeutics. iPSCs are the cells which are reprogrammed from somatic cells using different transcription factors. iPSCs possess unique properties of self renewal and differentiation to many types of cell lineage. Hence could replace the use of embryonic stem cells (ESC), and may overcome the various ethical issues regarding the use of embryos in research and clinics. Overwhelming responses prompted worldwide by a large number of researchers about the use of iPSCs evoked a large number of peple to establish more authentic methods for iPSC generation. This would require understanding the underlying mechanism in a detailed manner. There have been a large number of reports showing potential role of different molecules as putative regulators of iPSC generating methods. The molecular mechanisms that play role in reprogramming to generate iPSCs from different types of somatic cell sources involves a plethora of molecules including miRNAs, DNA modifying agents (viz. DNA methyl transferases), NANOG, etc. While promising a number of important roles in various clinical/research studies, iPSCs could also be of great use in studying molecular mechanism of many diseases. There are various diseases that have been modeled by uing iPSCs for better understanding of their etiology which maybe further utilized for developing putative treatments for these diseases. In addition, iPSCs are used for the production of patient-specific cells which can be transplanted to the site of injury or the site of tissue degeneration due to various disease conditions. The use of iPSCs may eliminate the chances of immune rejection as patient specific cells may be used for transplantation in various engraftment processes. Moreover, iPSC technology has been employed in various diseases for disease modeling and gene therapy. The technique offers benefits over other similar techniques such as animal models. Many toxic compounds (different chemical compounds, pharmaceutical drugs, other hazardous chemicals, or environmental conditions) which are encountered by humans and newly designed drugs may be evaluated for toxicity and effects by using iPSCs. Thus, the applications of iPSCs in regenerative medicine, disease modeling, and drug discovery are enormous and should be explored in a more comprehensive manner.

/pdf/induced-pluripotent-stem-cells-applications-in-regenerative-4jc4rvqncz.pdf

Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery

Induced Pluripotent Stem Cells (iPSCs) are self renewable and can differentiate to different types of adult cells, which has shown great promises in the field of regenerative medicine. iPSCs are reprogrammed from human somatic cells through ectopic expression of various transcription factors viz. Oct4, Sox2, Klf4, and c-Myc (OSKM). This novel technology enables derivation of patient specific cells, which possess a potential cure for many diseases. During the last decade, significant progresses have been achieved in enhancing the reprogramming efficiency, safety of iPSCs derivation, development of different delivery techniques by various research groups. Nevertheless, it is important to resolve and define the mechanism underlying the pluripotent stem cells. Major bottleneck which arises during iPSCs generation is the availability of source material (cells/tissues), difficulty to deliver transcription factors with no aberrant genetic modifications and limited reprogramming efficiency. Reprogramming may be achieved by employing different cocktails with number of different transcription factors, application of miRNA and some small molecules such as (Valproic acid, CHiR99021, Sodium butyrate, Vitamin C, Parnate etc). Similarly, various starting source materials have been demonstrated for iPSC based therapies including fibroblasts, cord blood, peripheral blood, keritinocytes, urine, etc., with their specific uses and limitations. Moreover, with the advent of many new reprogramming techniques, various direct delivery methods have been introduced such as using synthetic mRNA expressing pluripotent gene network has been shown to be an appropriate technique to deliver transcription factors and a dozen of small molecules which can replace transcription factors or enhance reprogramming efficiency. This article addresses the iPSCs technology mechanisms, progresses and current perspectives in the field.

Mechanism of Induction: Induced Pluripotent Stem Cells (iPSCs).

Stem cells are defined by their capabilities to self-renew and give rise to various types of differentiated cells depending on their potency. They are classified as pluripotent, multipotent and unipotent as demonstrated through their potential to generate the variety of cell lineages. While pluripotent stem cells may give rise to all types of cells in an organism, Multipotent and Unipotent stem cells remain restricted to the particular tissue or lineages. The potency of these stem cells can be defined by using a number of functional assays along with the evaluation of various molecular markers. These molecular markers include diagnosis of transcriptional, epigenetic and metabolic states of stem cells. Many reports are defining the particular set of different functional assays, and molecular marker used to demonstrate the developmental states and functional capacities of stem cells. The careful evaluation of all these methods could help in generating standard identifying procedures/markers for them.

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Describing the Stem Cell Potency: The Various Methods of Functional Assessment and In silico Diagnostics.

Ex vivo erythropoiesis methods are being developed for more than a decade now, and all the distinct types of stem cells (such as CD34+ HSCs, ESCs, IPSCs, and extensively proliferating erythropoietic progenitor cells) are defined to bear the potential for large scale RBC production shortly. The various regulating factors at different levels of RBCs production are being explored. Since most of the ex-vivo erythropoiesis protocols mimic the dogma followed by hematopoietic stem cells in vivo to give rise to mature RBCs which essentially deals with the intermediate stages of erythropoiesis such as burst forming unit-erythroid (BFU-E) and committed erythroid colony forming unit-erythroid (CFU-E). In vivo generation of erythroid progenitors (BFU-E/CFU-E) is essentially controlled by several factors including glucocorticoids, inflammation, and stress. Furthermore, regular production of functionally mature /transfusable units of RBCs is possible only through the coordinated regulation of terminal proliferation and differentiation of erythroid progenitors by external signals, such as erythropoietin, SCF, IL-3 and interaction to extracellular matrix protein(s) in a 3D culture system. We discuss these complex intracellular networks of coordinated factors and try to understand their molecular mechanism through gene regulation by transcription factors, and miRNAs that might be helpful in developing the optimal RBCs production protocols for commercial production.

Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation.

Blood transfusions are the mainstay in modern clinical modalities. The worldwide spread blood banks secure the supply of various blood components which solely depend upon voluntary paid/unpaid donor. According to the WHO reports present collections and supply of safe/adequate blood is inadequately distributed among economically developed and developing countries. Further, blood transfusions are prone to a number of risks/adverse reactions that may be imposed due to inefficient blood screening and processing methods and put extreme supply constraints, especially in the underdeveloped countries. In past few years stem cells from different sources are demonstrated to give rise to the significant amount of functionally mature erythrocytes, platelets and neutrophils by using a cocktail of growth factors under standard culture conditions. Therefore, a logistic approach to ex vivo manufacturing of erythrocytes (& other blood components) through modern tissue culture techniques has surfaced in the past few years. Ex vivo expansion of erythrocytes to generate transfusable units of clinical grade RBCs from a single dose of starting material (e.g. umbilical cord blood) has been well established. More and more information is coming from the various research groups showing the use of other potential sources (e.g. embryonic stem cells, induced pluripotent stem cells, and stress induced erythroid progenitors), different cocktails of growth factors, methodologies to get optimal yields of the functional RBCs/blood cells. One can imagine that with the advent of more defined methods for large-scale production of blood cells the existing system of donor derived blood supplies could be replaced in future. However, various regulating factors should essentially control the fate of large-scale production of RBCs including issues related to the quantity/quality and per unit cost of these products. These issues would be solved with the invention of more automated culture techniques such as the use of bioreactors and identification of more direct molecular agents regulating all phases of their ex vivo generation such as stem cell proliferation, differentiation, and maturation of final products. Recent studies have shown the possibilities of generating several hundred units of erythrocytes by using continuously feeding hollow fiber bioreactors along with other types of bioreactors. Even more, the functionality and acceptability of ex vivo cultured RBCs is demonstrated by their in-human transfusion which provides essential motivation to develop more sophisticated methods/culturing techniques to pave the way for commercial production of blood components at a feasible cost.

https://papers.ssrn.com/sol3/Delivery.cfm/SSRN_ID2986828_code2727604.pdf?abstractid=2986828&mirid=1&type=2

Manisha Kalsan

Papers

Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery

Mechanism of Induction: Induced Pluripotent Stem Cells (iPSCs).

Describing the Stem Cell Potency: The Various Methods of Functional Assessment and In silico Diagnostics.

Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation.

Blood Generation from Stem Cells: An Overview