Stem Cell Biology

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.

This perspective summarizes several important advances in hematopoietic stem cell (HSC) biology in the past few years and places these advances in the context of future directions in stem cell research. The potential utility of stem cells for gene therapy, tissue engineering, and the treatment of neurological and other forms of disease is simply too significant to ignore, and yet our knowledge and ability to deliver these forms of therapy in a safe and efficacious manner will require additional advances in the understanding of the basic biology of stem cells.

This perspective summarizes several important advances in hematopoietic stem cell (HSC) biology in the past few years and places these advances in the context of future directions in stem cell research. The potential utility of stem cells for gene therapy, tissue engineering, and the treatment of neurological and other forms of disease is simply too significant to ignore, and yet our knowledge and ability to deliver these forms of therapy in a safe and efficacious manner will require additional advances in the understanding of the basic biology of stem cells.

The transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) is critical for the regulation of normal stem cells maintenance by establishing specific epigenetic landscape. We have previously shown that CBP/p300 acetyltransferase induces PLZF acetylation in order to increase its deoxynucleotidic acid (DNA) binding activity and to enhance its epigenetic function (repression of PLZF target genes). However, how PLZF is inactivated is not yet understood. In this study, we demonstrate that PLZF is deacetylated by both histone deacetylase 3 and the NAD+ dependent deacetylase silent mating type information regulation 2 homolog 1 (SIRT1). Unlike other PLZF-interacting deacetylases, these two proteins interact with the zinc finger domain of PLZF, where the activating CBP/p300 acetylation site was previously described, inducing deacetylation of lysines 647/650/653. Overexpression of histone deacetylase 3 (HDAC3) and SIRT1 is associated with loss of PLZF DNA binding activity ...

In our experiments with an in vitro culture of rat cardiac cells, we identified and described for the first time the phenomenon of intracellular development of CSCs in mature CMs with for mation of the "cell-in-cell structures" (CICSs). Recently, we have confirmed the reproducibil ity of our results and existence of this phenomenon in rats of different age groups, 1-year-old bull, adult mice and humans. Moreover, we demonstrated the 5-10 times increase in the amount of CICSs after exposure of in vitro cultures to hypoxia and acidosis, that is, these con ditions stimulate intracellular development of CSCs. Our data strongly suggest that transitory amplifying cells (TACs), which release from CICSs, are present as a very rare cell population in adult and old rats. Therefore, we assume that TACs are important for renewal of myocardium during ontogenesis. TACs should be considered as the major source of cells that can reduce myocardial damage in adult mammals with various pathologies of the cardiovascular system. In conclusion, precise and exhaustive analysis of the phenomenon of intracellular develop ment of CSCs, CICSs and TACs will pave the way for cell technologies of new generation in regenerative medicine. primary culture of myocardial cells, mature cardiac myocytes, resident cardiac stem cells, proliferation, differentiation, cell-in-cell structure, transitory amplifying cells The founder of the study of stem cells (SCs) in adult organism is A.A. Maximow, who in 1909 first used the term «stem cell» and proved the existence of pluripotent hematopoietic stem

The maintenance of optimal placental physiology holds paramount importance for ensuring both balanced fetal growth and development, as well as the overall wellbeing of the mother during pregnancy. This significance arises from the placenta's intricate role in facilitating the transfer of essential nutrients and other factors crucial for achieving successful pregnancy outcomes. By acting as a conduit for this exchange between the maternal and fetal circulatory systems, the placenta establishes and sustains a complex physiological equilibrium. Beyond its immediate functions in supporting pregnancy, the human placenta emerges as a noteworthy source of stem cells with substantial implications for regenerative medicine. Stem cells derived from the placenta offer unique advantages, including the ability to foster optimal fetal growth and development. Importantly, the placenta's role extends beyond its temporal function during pregnancy, as it serves as a reservoir of diverse types of stem cells. The unique origin of placental tissues, originating in the early stages of embryonic development, raises intriguing possibilities. It suggests that these tissues may house cells retaining the plasticity observed in early embryonic cells, potentially unlocking therapeutic applications in regenerative medicine. The accessibility of the placenta in abundant quantities further enhances its appeal, and the non-intrusive nature of harvesting stem cells from this organ positions placenta-derived stem cells as highly attractive candidates for various regenerative medicine applications. This combination of factors underscores the promising potential of utilizing placental resources to advance therapeutic interventions in the field of regenerative medicine. In this special edition, we present four articles that cover a wide range of topics related to placental physiology, intrauterine inflammation, and gestational exposure to perfluorooctanoic acid (PFOA). Two of the included studies assessed the efficacies of artificial placenta in the maintenance of preterm birth in ovine and pig models. The other two studies focused on analyzing the effect of PFOA exposure and intrauterine inflammation on placental development. The study by Usuda et al. demonstrates the effective preservation of key physiological and hemodynamic parameters for up to 2 weeks in extremely preterm ovine fetuses, utilizing an artificial placenta platform and pharmacological intervention. Notably, this was achieved

This study to establish the effects of COVID-19 on 150 patients diagnosed with type 2 diabetes mellitus (T2DM) via a questionnaire distributed in Baghdad, Iraq, in light of social economy and healthcare restrictions. The present study seeks to explore and determine clinical interventions and physiological reactions employing a cross-sectional research design. In the demographic aspects, clinical characteristics, immunological markers, blood glucose level, and liver enzymes markers and SARS-CoV-2 IgG ELISA were evaluated.
While comparing the result values of diabetic COVID patients with control non-diabetic COVID patients it was observed that the fasting blood glucose level was 180 ± 40mg/dl as compared to 120 ± 30mg/dl, p < 0.001, postprandial blood glucose level was 240 ± 50mg/dl as compared to 160 ± 40mg/dl p < 0.01 and HbA1 Diabetic patients’ liver enzymes of ALT and AST were significantly higher than normal denoting that diabetes affects the liver. The percentage of hospitalized patients, those intubated in the ICU, those requiring mechanical ventilation, and those deaths among diabetic patients were higher compared with the control group; 80% vs 50%, p < 0. 001; 30% vs 15%, p < 0. 05; 25% vs 10%, p < 0. 01; and 20% vs 5%, p < additionally, dysregulation in immune and metabolic parameters was observed among T2DM patients with COVID-19, including elevated levels of pro-inflammatory cytokines (IL-6: 55. 0 ± 29.9 pg/mL, TNF-α: Although TNF-α was increased it was still slightly lower than normal (mean value 76.0 ± 34.5 pg/mL) as well as IL-1-b (mean 23.9 ± 12.5 pg/mL) while the indicators of glycemic control were poor HbA1c (mean 8.7 ± 2.0%) and random glucose (mean 240.6 ± 78.3 Elevated liver enzymes (ALT: (ALP: 44.5 ± 21.9 U/L, AST: 38.9 ± 19.8 U/L, GGT: 54.1 ± 29.8 U/L) supported that there was hepatic stress. Clinical outcomes were also poor, with 80 % of the patients being hospitalized, 34%; intensive care unit admission was required for 19 % of the patients. 7% were admitted to the ICU and 14.7% mortality. Non-survivors exhibited significantly higher inflammatory markers and poorer glycemic control compared to survivors (HbA1c: IL-10 concentration was higher in the study group (10.0 ± 2. 3% vs. 8.3 ± 1. 8%); IL-6 (80.7 ± 33.8 pg/mL vs. 48.6 ± 27.8 pg/mL) and TNF-α (116.4 ± 38.5 pg/mL vs. 68.1 ± 30.9 pg/mL).

Spermatogonial stem cells (SSCs) also known as germ stem cells (GSCs) are the basis of spermatogenesis process in the testis. Furthermore, they are also valuable cells with different applications in developmental biology, transgenesis technology, and clinic. Understanding the new findings related to the cell and molecular biology of SSCs and the methods for isolation and maintenance of these cells are important and essential for their applications in medicine to treat some infertility problems and also in biotechnology to produce transgenic animals. The present review was conducted to describe the cell and molecular basis of development, self-renewal, and differentiation of mammalian and poultry SSCs in vivo (natural niche) and in vitro. Moreover, this study represents specific molecular markers to characterize SSCs. We also introduce methods to isolate, cultivate and enrich these cells, which are important for their applications. Finally, the significance of SSCs in different field...

Flow cytometric analysis is a valuable technique for identification and characterization of cells. However, flow cytometric analysis of autofluorescent cells requires careful consideration. In particular, the outcome of fluorescence compensation in the presence of significant autofluorescence poses a challenge to the interpretation of multicolour flow cytometric data. In this paper, we explain the mathematical basis of fluorescence compensation and the effects of autofluorescence, both in theory and applied to mesenchymal stem cells, which are notoriously autofluorescent. We illustrate how failure to understand the consequences of compensation can easily lead to critical errors in data interpretation, particularly when autofluorescence is involved. In the process, the common pitfalls of flow cytometric analysis of mesenchymal stem cells are presented, as are the simple measures necessary to avoid them. Specifically, the counterintuitive concept of "negative fluorescence values" is explained and exemplified, and the phenomenon of "population broadening" is addressed. Researchers must be acutely aware of the effects of compensation on the positioning of cells. We recommend always displaying data in biexponential or logicle transformations and advice to include fluorescence-minus-one controls to establish thresholds of positivity.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC) or two (OCT4, KLF4; hiPSC2F-NSC) reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB) or four reprogramming factors (hiPSC4F-FIB). After four weeks of coculture with ...

Three induced pluripotent stem cells (iPSC) clones NCCSi007-A, NCCSi007-B and NCCSi007-C were generated from CD4 + T cells of a 38 years old male patient suffering from liver cirrhosis-alcoholic and minimal hepatic encephalopathy of Indian origin. The CD4 + T cells of the patient were reprogrammed using integration free, Sendai viral vector system. Each of the three iPSC clones showed high alkaline phosphatase (ALP) activity, expressed pluripotency markers OCT4, SOX2, NANOG, KLF4, SSEA-4, TRA-1-60, showed normal male karyotype (46, XY) and exhibited multi-lineage differentiation. Resource table Unique stem cell lines identifier NCCSi007-A, NCCSi007-B and NCCSi007-C Alternative names of stem cell lines NCCSi007-A (DYPM1-M2), NCCSi007-B (DYPM1-M3) and NCCSi007-C (DYPM1-M4)

Urine samples were collected from three patients (2 males, 1 female) with clinically diagnosed Attention Deficit Hyperactivity Disorder (ADHD) according to DSM-5 criteria using semi-structured interviews (K-SADS adapted for adults) by a trained psychiatrist. Urine epithelial cell lines were established and expanded for subsequent reprogramming procedure. Induced pluripotent stem cells (iPSCs) were derived using integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, which includes Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Urine sample was collected from a 29-year-old male patient with an early onset form of DSM-5 obsessive-compulsive disorder (OCD), comorbid body dysmorphic and excoriation (skin-picking) disorders, and a positive family history for OCD. Urine cell line was established and expanded for the reprogramming procedure. Induced pluripotent stem (iPS) cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, which includes Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Skin biopsies were collected from three male patients (age 45, 44 and 44) with clinically diagnosed schizophrenia. The patients were diagnosed according to DSM-5 criteria by a trained psychiatrist. Dermal fibroblast cell lines were established and expanded for subsequent reprogramming procedures. Induced pluripotent stem (iPS) cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, containing Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.

We describe the internal organization of murine embryoid bodies (EBs) in terms of the structures and cell types formed as Oct4 expression becomes progressively lost. This is done by making the EBs from iPS cells carrying an inducible and permanent Oct4 reporter (Oct4-MerCreMer;mTmG). When these EBs are treated with tamoxifen, the Oct4 expressing cells switch from a red to a green fluorescence color, and this is maintained thereafter by their progeny. We show that there is no specific pattern in which Oct4 is downregulated, rather it appears to be spatially random. The earliest cells to lose Oct4 expression are internal and stain positive for -fetoprotein (AFP) indicating that they are visceral endoderm. However, GATA4, characteristic of primitive

Toward programmed therapeutics Advances in synthetic biology are enabling the development of new gene and cell therapies. Kitada et al. review recent successes in areas such as cancer immunotherapy and stem cell therapy, point out the limitations of current approaches, and describe prospects for using synthetic biology to overcome these challenges. Broader adoption of these therapies requires precise, context-specific control over cellular behavior. Gene circuits can be built to give sophisticated control over cellular behaviors so that therapeutic functions can, for example, be programmed to activate in response to disease biomarkers. Science , this issue p. eaad1067

Developmental lung biology is a field that has the potential for significant human impact: lung disease at the extremes of age continues to cause major morbidity and mortality worldwide. Understanding how the lung develops holds the promise that investigators can use this knowledge to aid lung repair and regeneration. In the decade since the "molecular embryology" of the lung was first comprehensively reviewed, new challenges have emerged-and it is on these that we focus the current review. Firstly, there is a critical need to understand the progenitor cell biology of the lung in order to exploit the potential of stem cells for the treatment of lung disease. Secondly, the current familiar descriptions of lung morphogenesis governed by growth and transcription factors need to be elaborated upon with the reinclusion and reconsideration of other factors, such as mechanics, in lung growth. Thirdly, efforts to parse the finer detail of lung bud signaling may need to be combined with broader consideration of overarching mechanisms that may be therapeutically easier to target: in this arena, we advance the proposal that looking at the lung in general (and branching in particular) in terms of clocks may yield unexpected benefits.

Cell differentiation in Entamoeba and Giardia exhibit a great deal of similarity. Unfortunately, both protists were studied over the years mostly under axenic conditions, disregarding the natural micro-ecology of host intestine. Using hypoxic culture sediments with oxygen consuming bacteria (OCB niches) we discovered in E. invadens an ancient stem cell system that shows the same basal mechanisms for stemness that exist in highly developed stem cell systems too. The ancient stem cells of E. invadens (AnSC) have all standards of the stem cell definition (self renewing and quiescent cells, glycolytic metabolism, stem cell plasticity, reprogramming and induced totipotency recovery, and both induced and autonomous terminal differentiation) indicating that stem cells were developed by the anaerobe proterozoic LECA-as stages of its life cycle-then diverged into all eukaryotes. The more protist stem cell systems we can dechipher the more we will learn about the ancestral protolineage, its cell line hierarchy and basal mechanism of stemness. We assume that Giardia could be a good model system to expand protist stem cell biology. We found in Giardia literature evidence suggesting self renewal and quiescence, plasticity and other stem cell characteristics. The present study is a reinterpretation of Giardia's life cycle in the light of current stem cell knowledge. It is hoped this reinterpretation stimulates new developments.

The aim and the background of this paper was to investigate how ancient primitive eukaryotes evolved to the successful parasites they are today. In preparing this work, the most significant literature of the last years has been studied. We expand it by the results of our own research. Results and conclusion: Anaerobic single-celled eukaryotes, such as Entamoeba invadens and Giardia lamblia, became successful invasive pathogens by using mechanisms inherited from the common ancestor (LECA). As described in previous papers pathogen protists have a surprising stem cell network (ancient protolineage) controlled by intrinsic and extrinsic mechanisms of cell conversion and differentiation inherited from the ancestor. Mechanisms leading to pathogenicity were not acquired after the organisms became parasitic, they were present before the single-celled organisms switched to parasitism. Only organisms possessing an extended ancestral stem cell system capable of switching between most oxidative (MO) and most hypoxic (MH) niches by changing metabolic pathways and antigenicity could develop into successful invasive pathogens like Entamoeba or Giardia. Related organisms not conserving all ancestral features evolved to become luminal commensals or free-living protists.

Ecosystems and Stem Cells Niches The life cycle of pathogenic amoebae begins in the ileo-jejunal region of the human duodendum, where ingested cysts hatch. Amoebic cells passing the ileo-cecal valve (ileocolic orifice) colonize the colon, in particular the cecum and the sigmorectal region. Entamoeba invadens living in reptiles is in a biological sense the close relative of Entamoeba histolytica. It has played a key role in deciphering the amoebic life cycle and stem cell lineages of pathogenic and less pathogenic amoebae. In this author's opinion the closely related stem cell lineages of E. invadens, E. histolytica and E. dispar are inherited from the common eukaryotic ancestor and therefore closely related [1]. They are each subjected to the oxygen gradients of the intestine. The key role of oxygen gradients could be clarified for the first time in cultures sediments with oxygen consuming bacteria (OCB cultures) [1]. OCB grown amoebae regulated by oxygen gradients reveal much more about the stem cell biology

Entamoeba invadens is not the secondarily reduced and simplified eukaryote unrelated to a cenancestor as thought by some protist researchers. On the contrary, E. invadens is a single-celled eukaryote with a complex life cycle comprised of stem cells and stem cell lines. It contains hypoxic and oxygenic life cycle stages revealing to us details about the living conditions of LECA at the time of Unikonts' and Excavata's divergence. At the end of the super group evolution are protists such as Entamoeba and Giardia and metazoans such as Hydra and humans. Stem cells in Entamoeba and humans are controlled by the same basal mechanisms. At the time of super group divergence LECA had an anaerobic metabolism similar to that of Entamoeba; it contains non-aerobic mitochondria, different metabolic pathways using oxygen or not, anti-oxidative defense mechanisms against oxygen and a life cycle organized with a stem cell lineage. LECA evolved in the Proterozoic age from a unique vegetative cell type living in strict hypoxic environments (temporarily protected by a cyst wall) to a more and more complex cell system capable of living and proliferating in a wide range of hypoxic and moderate oxygenic niches. In conditions of alternating oxygen-, nitrogen-and nutrient-cycles LECA developed the characteristics of a true stem cell protolineage and transferred them to protist divergents and early animals. Without this ancestral heritage, protists such as E. invadens would not be able to become pathogens. They would live in a commensal relationship with their host. The metabolic anaerobe LECA is the common cenancestor of all eukaryotic stem cell lineages.

In Entamoeba stem cells, oxygen availability regulates proliferation and differentiation. In the past Entamoebae were categorized as "micro-aerophile" organisms. Later, they were considered to be anaerobes because they perform an anaerobic/ fermentative metabolism and possess mitosomes/ dehydrogenosomes instead of mitochondria. Since the discovery of the stem cell lineage of Entamoeba it is known that the pathogen amoebae have a primary multipotent stem cell line (p-SRL) that converted in two progenitor stem cell lines of reduce potency producing a single type of differentiate cell (MAS or MAT) each. Both progenitor lines preferentially colonize the cecum that provides usually an oxygen gradient of <1%-5.5% O 2 content. The s-SRL line is an oxygenic cell line of limited self-renewal residing in the upper oxygenic zones; it proliferates by fast cycling and differentiates to ATD cysts by intrinsic mechanisms of terminal differentiation via committed MAS cells. MAS cells are precursor cells for natural encystment and form ATD cysts. The other cell line is the unipotent t-SRL line living in all gradient zones. It switches from fast to slow cycling and back again and differentiate non-committed MAT cells. Hypoxia <1% O 2 amplifies the t-SRL line by complete self renewal (symmetric cell fate) preserving the undifferentiated hypoxic cell state (ISH cells). Declining hypoxia reconstitutes the slow cycling t-SRL line. Oxygen depletion represses terminal differentiation: first, it forces the s-SRL line to converse into a non-encysting t-SRL line and secondly, it interrupts precursor cell progression through the developmental cell cycle.

Negative bHLH transcription factor Hes1 can inhibit neural stem cells (NSCs) from precocious neurogenesis through repressing proneural gene expression; therefore, sustenance of Hes1 expression is crucial for NSC pool maintenance. Here, we find that Ids, the dominant-negative regulators of proneural proteins, are expressed prior to proneural genes and share an overlapping expression pattern with Hes1 in the early neural tube of chick embryos. Overexpression of Id2 in the chick hindbrain upregulates Hes1 expression and inhibits proneural gene expression and neuronal differentiation. By contrast, Hes1 expression decreases, proneural gene expression expands and neurogenesis occurs precociously in Id1;Id3 double knockout mice and in Id1-3 RNAi-electroporated chick embryos. Mechanistic studies show that Id proteins interact directly with Hes1 and release the negative feedback autoregulation of Hes1 without interfering with its ability to affect other target genes. These results indicate that Id proteins participate in NSC maintenance through sustaining Hes1 expression in early embryos.

Regenerative dentistry represents an attractive multidisciplinary therapeutic approach that complements traditional restorative/surgery techniques and benefits from recent advances in stem cell biology, molecular biology, genomics and proteomics. Materials science is important in such advances to move regenerative dentistry from the laboratory to the clinic. The design of novel nanostructured materials, such as biomimetic matrices and scaffolds for controlling cell fate and differentiation, and nanoparticles for diagnostics, imaging and targeted treatment, is needed. The combination of nanotechnology, which allows the creation of sophisticated materials with exquisite fine structural detail, and stem cell biology turns out to be increasingly useful in regenerative medicine. The administration to patients of dynamic biological agents comprising stem cells, bioactive scaffolds and/or nanoparticles will certainly increase the regenerative impact of dental pathological tissues. This ove...

Background: Progenitor cells exist in most tissues and body fluids. Synovial fluid chondroprogenitor cells have been described in several species; however, the specific phenotypic characteristics of these cells have not been defined. This study addressed the impacts of joint location and donor age variation on synovial fluid chondroprogenitor numbers, and determined whether synovial fluid chondroprogenitors express hypertrophic characteristics or a non-endochondral phenotype in chondrogenic culture. Methods: Synovial fluid was aspirated from the joints of healthy adult horses, and cells in the aspirates were expanded through two monolayer passages. The number of colony-forming units (CFU) in each aspirate was assessed after 14 days. Population doublings (PD) and PD times were calculated during the subsequent two passages. Passage 3 cells were used to determine osteogenic and adipogenic capacities, or were transferred to pellet cultures in chondrogenic medium containing TGF-β1 or BMP-2. Bone marrowderived MSCs and primary articular chondrocytes were cultured under similar conditions to provide reference values for chondrogenesis. Chondrogenesis was assessed by measuring collagen type II protein and sulfated glycosaminoglycan secretion, expression of chondrogenic mRNAs, and induction of ALP activity. Results: CFU counts varied considerably, but the majority of aspirates contained <50 CFU/ml. PDs were consistently between 2.0 and 3.0 during both passages. PD times varied from 0.2-0.4 days. Proliferation was not significantly influenced by anatomical location or donor age. Equine synovial fluid progenitors expressed osteogenic, adipogenic and chondrogenic phenotypes under appropriate conditions. Under chondrogenic conditions, synovial fluid cells increased collagen and aggrecan mRNA expression to levels comparable to bone marrow MSCs, although collagen type II protein secretion was less than half that of articular chondrocytes. In contrast to bone marrow MSCs, synovial fluid chondroprogenitors did not express collagen type X or increase ALP activity in response to TGF-β1 or BMP-2. Consistent with these observations, Runx2 and Mef2C expression in synovial fluid chondroprogenitors was 20-40 fold lower than in bone marrow MSCs. Conclusions: Synovial fluid chondroprogenitors are capable of robust non-hypertrophic chondrogenesis, whether stimulated by TGF-β1 or by BMP-2. These results indicate that synovial fluid cells are phenotypically suitable for articular cartilage repair/regeneration, although strategies to accelerate cell proliferation and synthesize a functional cartilage matrix in vivo will be required for clinically feasible cellbased therapies.

Adult skeletal muscle is a postmitotic tissue with an enormous capacity to regenerate upon injury. This is accomplished by resident stem cells, named satellite cells, which were identified more than 50 years ago. Since their discovery, many researchers have been concentrating efforts to answer questions about their origin and role in muscle development, the way they contribute to muscle regeneration, and their potential to cell-based therapies. Satellite cells are maintained in a quiescent state and upon requirement are activated, proliferating, and fusing with other cells to form or repair myofibers. In addition, they are able to self-renew and replenish the stem pool. Every phase of satellite cell activity is highly regulated and orchestrated by many molecules and signaling pathways; the elucidation of players and mechanisms involved in satellite cell biology is of extreme importance, being the first step to expose the crucial points that could be modulated to extract the optimal ...

Background: The c.G6055A (p.G2019S) mutation in leucine-rich repeat kinase 2 (LRRK2) is the most prevalent genetic cause of Parkinson's disease (PD). CRISPR/Cas9-mediated genome editing by homology-directed repair (HDR) has been applied to correct the mutation but may create small insertions and deletions (indels) due to double-strand DNA breaks. Adenine base editors (ABEs) could convert targeted A•T to G•C in genomic DNA without double-strand breaks. However, the correction efficiency of ABE in LRRK2 c.G6055A (p.G2019S) mutation remains unknown yet. This study aimed to compare the mutation correction efficiencies and off-target effects between HDR and ABEs in induced pluripotent stem cells (iPSCs) carrying LRRK2 c.G6055A (p.G2019S) mutation. Methods: A set of mutation-corrected isogenic lines by editing the LRRK2 c.G6055A (p.G2019S) mutation in a PD patient-derived iPSC line using HDR or ABE were established. The mutation correction efficacies, off-target effects, and indels between HDR and ABE were compared. Comparative transcriptomic and proteomic analyses between the LRRK2 p.G2019S iPSCs and isogenic control cells were performed to identify novel molecular targets involved in LRRK2-parkinsonism pathways. Results: ABE had a higher correction rate (13/53 clones, 24.5%) than HDR (3/47 clones, 6.4%). Twenty-seven HDR clones (57.4%), but no ABE clones, had deletions, though 14 ABE clones (26.4%) had off-target mutations. The corrected isogenic iPSC-derived dopaminergic neurons exhibited reduced LRRK2 kinase activity, decreased phosphoα-synuclein expression, and mitigated neurite shrinkage and apoptosis. Comparative transcriptomic and proteomic analysis identified different gene expression patterns in energy metabolism, protein degradation, and peroxisome proliferator-activated receptor pathways between the mutant and isogenic control cells. Conclusions: The results of this study envision that ABE could directly correct the pathogenic mutation in iPSCs for reversing disease-related phenotypes in neuropathology and exploring novel pathophysiological targets in PD.

A novel bone tissue-engineering construct was developed by using poly(ɛ-caprolactone) (PCL)-macrochanneled scaffolds combined with stem cell-seeded collagen hydrogels and then applying flow perfusion culture. Rat mesenchymal stem cells (MSCs) were loaded into collagen hydrogels, which were then combined with macrochanneled PCL scaffolds. Collagen hydrogels were demonstrated to provide favorable growth environments for MSCs and to foster proliferation. Cell number determination identified retention of substantially fewer (50-60%) cells when they were seeded directly onto macrochanneled PCL than of cells engineered within collagen hydrogels. Additionally, the cells actively proliferated within the combined scaffold for up to 7 days. MSC-loaded collagen-PCL scaffolds were subsequently cultured under flow perfusion to promote proliferation and osteogenic differentiation. Cells proliferated to levels significantly higher in flow perfusion culture than that under static conditions during ...

The degree to, and the mechanisms through, which stem cells are able to build, maintain, and heal the body have only recently begun to be understood. Much of the stem cell&#39;s power resides in the release of a multitude of molecules, called stem cell released molecules (SRM). A fundamentally new type of therapeutic, namely &quot;systems therapeutic&quot;, can be realized by reverse engineering the mechanisms of the SRM processes. Recent data demonstrates that the composition of the SRM is different for each type of stem cell, as well as for different states of each cell type. Although systems biology has been successfully used to analyze multiple pathways, the approach is often used to develop a small molecule interacting at only one pathway in the system. A new model is emerging in biology where systems biology is used to develop a new technology acting at multiple pathways called &quot;systems therapeutics&quot;. A natural set of healing pathways in the human that uses SRM is in...

The biopharmaceutical relevance of producing plasmid DNA at large scale has increased steadily over the years due to the development of a growing number of direct and indirect applications. Be it as biological drugs or as starting materials, plasmids are pervasive across the gene and cell therapy industry of today. With hundreds of biopharmaceutical companies using plasmids in the clinical development of their products, plasmid manufacturing is starting to emerge as a key bottleneck. This commentary provides an overview of the uses of plasmids, discusses manufacturing challenges, and hints at what the future may bring.

Yes-associated protein (YAP) is an important transcriptional coactivator in the Hippo signaling pathway. Using CRISPR/Cas9 technology, we established a stable YAP-knockdown (YAP-KD) induced pluripotent stem cell (iPSC) from the MUSIi012-A cell line. The YAP-KD iPSC MUSIi012-A-2 maintained the pluripotent phenotype, the ability to differentiate into all three embryonic germ layers, and it maintained the normal karyotype. Resource table Unique stem cell line identifier MUSIi012-A-2 Alternative name(s) of stem cell line YAP-KD cell line

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.

Adult skeletal muscle is a postmitotic tissue with an enormous capacity to regenerate upon injury. This is accomplished by resident stem cells, named satellite cells, which were identified more than 50 years ago. Since their discovery, many researchers have been concentrating efforts to answer questions about their origin and role in muscle development, the way they contribute to muscle regeneration, and their potential to cell-based therapies. Satellite cells are maintained in a quiescent state and upon requirement are activated, proliferating, and fusing with other cells to form or repair myofibers. In addition, they are able to self-renew and replenish the stem pool. Every phase of satellite cell activity is highly regulated and orchestrated by many molecules and signaling pathways; the elucidation of players and mechanisms involved in satellite cell biology is of extreme importance, being the first step to expose the crucial points that could be modulated to extract the optimal ...

A conference, &quot;Stem Cells and Cell Therapies in Lung Biology and Lung Diseases,&quot; was held July 25 to 28, 2011 at the University of Vermont to review the current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy and ex vivo bioengineering approaches for lung diseases. These are rapidly expanding areas of study that provide further insight into and challenge traditional views of mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, to discuss and debate current controversies, and to identify future research directions and opportunities for basic and translational research in cell-based therapies for lung diseases. The goal of this article, which accompanies the formal conference report, is to provide a comprehensive review of the published literature in lung regenerative medicine from the l...

Human pluripotent stem cells (hPSCs) were conventionally cultured on feeder cells that are isolated from mouse embryonic fibroblast (MEF). However, these culture components could contaminate the hPSCs and can limit the application of hPSCs for clinical use. On the other hand, we demonstrated that exogenous basic fibroblast growth factor (bFGF) could be omitted from the hPSC culture media if we used the suitable feeder cells. We also showed that although hPSCs can proliferate on the feeder-free culture system, however, genetic instability of hPSCs has been reported in such environment. Feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. We proposed the use of human cord blood-derived serum (hUCS) and showed a positive effect on culture of mesenchymal stem cells. The results showed that human foreskin fibroblasts (HFFs) cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, short population doubling times, high proliferation rates, and normal karyotypes after prolonged culture. These studies of hPSC xeno-free culture have been growing in both basic research and clinical trial. The data regarding the current clinical trials of using hPSCs convince the researchers not only about the possibility of application of hPSCs for cell-based therapy, but also the quality of established hPSC lines. Most of the hPSC lines that were published in the literature and registered in the National Institute of Health (NIH), hPSCreg of the European Union are not Good Manufacturing Practice (GMP) grade cell lines. Since one of the goals of using hPSCs is therapeutic purpose, GMP for derivation, cultivation, and handling the hPSCs are required. This chapter also reviews the state-of-the-art xeno-free culture system of hPSCs in the respect of future clinical applications.

Rudolf Jaenisch is a Professor of Biology at Massachusetts Institute of Technology, a founding member of the Whitehead Institute for Biomedical Research and the current president of the International Society for Stem Cell Research (ISSCR). His contributions to the stem cell field span from making the first transgenic mouse to seminal advances in the reprogramming field, and much more. In recognition of his pioneering research leading to induced pluripotency, he recently received the 2015 March of Dimes Prize in Developmental Biology. At the recent Keystone Meeting on ‘Transcriptional and Epigenetic Influences on Stem Cell States’ in Colorado, we had the opportunity to talk to him about his life and work.

Human mesenchymal stem cells (MSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications. Exposure of MSCs to physical stimuli favors early and rapid activation of the tissue repair process. In this study we investigated the in vitro effects of pulsed electromagnetic field (PEMF) treatment on the proliferation and osteogenic differentiation of bone marrow MSCs (BM-MSCs) and adipose-tissue MSCs (ASCs), to assess if both types of MSCs could be indifferently used in combination with PEMF exposure for bone tissue healing. We compared the cell viability, cell matrix distribution, and calcified matrix production in unstimulated and PEMF-stimulated (magnetic field: 2 mT, amplitude: 5 mV) mesenchymal cell lineages. After PEMF exposure, in comparison with ASCs, BM-MSCs showed an increase in cell proliferation (p < 0.05) and an enhanced deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and-III collagens (p < 0.05). Calcium deposition was 1.5-fold greater in BM-MSC-derived osteoblasts (p < 0.05). The immunofluorescence related to the deposition of bone matrix proteins and calcium showed their colocalization to the cell-rich areas for both types of MSC-derived osteoblast. Alkaline phosphatase activity increased nearly 2-fold (p < 0.001) and its protein content was 1.2-fold higher in osteoblasts derived from BM-MSCs. The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed up-regulated transcription specific for bone sialoprotein, osteopontin, osteonectin, and Runx2, but at a higher level for cells differentiated from BM-MSCs. All together these results suggest that PEMF promotion of bone extracellular matrix deposition is more efficient in osteoblasts differentiated from BM-MSCs.

Urine samples were collected from three patients (2 males, 1 female) with clinically diagnosed Attention Deficit Hyperactivity Disorder (ADHD) according to DSM-5 criteria using semi-structured interviews (K-SADS adapted for adults) by a trained psychiatrist. Urine epithelial cell lines were established and expanded for subsequent reprogramming procedure. Induced pluripotent stem cells (iPSCs) were derived using integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, which includes Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPSC clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson’s disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G&gt;C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy prior to generating footprint-free i...

The data consist of (1) iridophore counts in WT or tfec mutant embryos at 3 dpf (Fig. 3F); (2) iridophore counts from tfec mutants injected with tfec cDNA construct or uninjected controls (Fig. 3K); (3) iridophore counts from WT embryos injected with various control and tfec-expression plasmids, and uninjected controls (Fig. 3M); (4) melanocyte counts in tfec mutants compared to WT siblings at 30 hpf and 4 dpf (Fig. 4I,L).

Cells actively sense and process mechanical information that is provided by the extracellular environment to make decisions about growth, motility and differentiation. It is important to understand the underlying mechanisms given that deregulation of the mechanical properties of the extracellular matrix (ECM) is implicated in various diseases, such as cancer and fibrosis. Moreover, matrix mechanics can be exploited to program stem cell differentiation for organ-on-chip and regenerative medicine applications. Mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology, bioengineering and biophysics. Here we provide an introductory overview of the key players important to cellular mechanobiology, taking a biophysical perspective and focusing on a comparison between flat versus three dimensional substrates.

Krueppel-like factor 15 (KLF15) is abundantly expressed in liver, kidney, and muscle, including myocardium. In the adult heart KLF15 is important to maintain homeostasis and to repress hypertrophic remodeling. We generated a homozygous hESC KLF15 knockout (KO) line using paired CRISPR/Cas9n. KLF15-KO cells maintained full pluripotency and differentiation potential as well as genomic integrity. We demonstrated that KLF15-KO cells can be differentiated into morphologically normal cardiomyocytes turning them into a valuable tool for studying human KLF15-mediated mechanisms resulting in human cardiac dysfunction.

A conference, &quot;Stem Cells and Cell Therapies in Lung Biology and Lung Diseases,&quot; was held July 25 to 28, 2011 at the University of Vermont to review the current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy and ex vivo bioengineering approaches for lung diseases. These are rapidly expanding areas of study that provide further insight into and challenge traditional views of mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, to discuss and debate current controversies, and to identify future research directions and opportunities for basic and translational research in cell-based therapies for lung diseases. The goal of this article, which accompanies the formal conference report, is to provide a comprehensive review of the published literature in lung regenerative medicine from the l...

Background: Limbal epithelial stem cells (LESC) have great potential in treating the blindness caused by corneal damage. LESC are maintained in stem cell niche called Palisade of Vogt. Limbal stromal (LS) cells are critical component of LESC niche and help in their self renewal. These cells resemble mesenchymal stromal/stem cells with multilineage differentiation potential. However little is known about their gene expression profile compared to MSC derived from various sources. Methods: In this study, we compared the gene expression profile of LS cells expanded in two different culture conditions: basal media with bFGF, LIF and matrigel (LS-matrigel), as well as basal media with 10% FBS (LS-FBS). In addition, gene expression profile of LS-FBS cells were compared to bone marrow, adipose-derived MSC and foreskin fibroblasts. Total RNA was extracted from various cell types upon achieving confluency and subjected to microarray experiments (Agilent platform) using Human GE 8x60k gene chips. Data analysis was done with GeneSpring software. Results: LS cells cultured in matrigel system showed upregulation of 871 genes as compared to LS-FBS and 58 genes were consistently differentially expressed in LS-FBS as compared to other cell types. Despite many long intergenic non-coding RNA and function unknown genes, differentially expressed genes represent gene ontology for cell signaling molecules including various growth factors, cell metabolism and extracellular matrix components for various biological processes. Samples from the same source were closely clustered by hierachical clustering analysis. Conclusions: The two culture conditions used in the study affected the gene expression profile of LS cells significantly. The LS cells showed distinct molecular signature as compared to MSC from various other sources. This comparative study will help in understanding the limbal stem cell niche biology and the novel set of genes could be used as biomarkers for LS cells.