Stages Of Cell Differentiation Research Articles

Identification of the Differentiation Stages of Living Cells from the Six Most Immature Murine Hematopoietic Cell Populations by Multivariate Analysis of Single-Cell Raman Spectra.

Efforts to expand hematopoietic stem and progenitor cells (HSPCs) in vitro are motivated by their use in the treatment of leukemias and other blood and immune system diseases. The combinations of extrinsic cues within the hematopoietic stem cell (HSC) niche that lead to HSC fate decisions remain unknown. New noninvasive and location-specific techniques are needed to enable identification of the differentiation stages of individual hematopoietic cells on biomaterial microarray screening platforms that minimize the usage of rare HSCs. Here, we show that a combination of Raman microspectroscopy and partial least-squares discriminant analysis (PLS-DA) enables the location-specific identification of individual living cells from the six most immature hematopoietic cell populations, HSC, multipotent progenitor (MPP)-1, MPP-2, MPP-3, common myeloid progenitor, and common lymphoid progenitor. Better than 90% accuracy was achieved. We show that the accuracy of this differentiation stage identification was based on spectral features associated with cell biochemistries. This work establishes that PLS-DA can capture the subtle spectral variations between as many as six closely related cell populations in the presence of potentially significant within-population spectral variation. This noninvasive approach can be used to screen HSC fate decisions elicited by extrinsic cues within biomaterial microarray screening platforms.

Effects of l-Tryptophan and 1,25-Dihydroxycholecalciferol on Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Isolated from the Compact Bones of Broilers and Layers

Poultry is vulnerable to bone problems throughout their lives or production period due to rapid growth in broilers and the active laying cycle in layers. The calcium-sensing receptor (CaSR) is important in calcium and bone metabolism. The objective of this study was to investigate the effect of the CaSR ligand (l-Trp) and 1,25-dihydroxycholecalciferol (1,25OHD3) on the regulation of proliferation and osteogenic differentiation of chicken mesenchymal stem cells (MSCs) isolated from the compact bones of 14-day-old Ross 308 chickens and Dekalb pullets, which can provide cell-based evidence for the prevention or alleviation of skeletal disorders in the poultry industry. First, the dose- (0, 0.5, 1, 2, 5, 10, and 15 mM) and time-effects (0, 7, and 14 days) of l-Trp on the proliferation and osteogenic differentiation in chicken MSCs were investigated. The 5 mM l-Trp had a balanced effect between proliferation and osteogenic differentiation in broiler and layer MSCs when differentiated for 7 days. The broiler and layer MSCs were then treated with (1) osteogenic medium, osteogenic medium supplemented with (2) 1 nM 1,25OHD3, (3) 2.5 mM Ca2+, (4) 2.5 mM Ca2+ + 5 mM l-Trp and (5) 2.5 mM Ca2+ + 5 mM l-Trp + 1 μM NPS-2143, separately for 7 days. Results showed that the 5 mM l-Trp significantly inhibited the proliferation of broiler and layer MSCs on day 7 (P < 0.05), but 1 nM 1,25OHD3 significantly promoted the proliferation of layer MSCs (P < 0.05). Only the 2.5 mM Ca2+ + 5 mM l-Trp group significantly increased the mineralization process during osteogenic differentiation (P < 0.05), and this treatment also significantly upregulated the mRNA expression of the vitamin D receptor (VDR), β-catenin, and osteogenesis genes in broiler MSCs (P < 0.05). The osteogenic differentiation process in layer MSCs was faster than that in broiler MSCs. In layer MSCs, Ca2+ alone significantly facilitated mineralization and ALP activity after 7-day osteogenic differentiation (P < 0.05). However, the 5 mM l-Trp significantly inhibited the differentiation and mineralization process by downregulating the mRNA expression of CaSR, VDR, β-catenin, and osteogenic genes (P < 0.05) in layer MSCs. Taken together, l-Trp and 1,25OHD3 can regulate proliferation and osteogenic differentiation in both broiler and layer MSCs depending on the dose, treatment time, and cell proliferation and differentiation stages.

Expression of Cell Cycle Markers and Proliferation Factors during Human Eye Embryogenesis and Tumorigenesis.

The expression pattern of the markers p19, Ki-67, MSX1, MSX2, PDL1, pRB, and CYCLINA2 was quantitatively and semiquantitatively analyzed in histologic sections of the developing and postnatal human eye at week 8, in retinoblastoma, and in various uveal melanomas post hoc studies by double immunofluorescence. The p19 immunoreactivity characterized retinal and/or choroidal cells in healthy and tumor tissues: expression was lower in the postnatal retina than in the developing retina and retinoblastoma, whereas it was high in epithelioid melanomas. Ki67 expression was high in the developing eye, retinoblastoma, and choroidal melanomas. MSX1 and MSX2 expression was similar in the developing eye and retinoblastoma, whereas it was absent in the postnatal eye. Their different expression was evident between epithelioid and myxoid melanomas. Similarly, PDL1 was absent in epithelioid melanomas, whereas it was highly expressed in developing and tumor tissues. Expression of pRB and CYCA2 was characteristic of developing and tumorous eye samples but not of the healthy postnatal eye. The observed expression differences of the analyzed markers correlate with the origin and stage of cell differentiation of the tissue samples. The fine balance of expression could play a role in both human eye development and ocular tumorigenesis. Therefore, understanding their relationship and interplay could open new avenues for potential therapeutic interventions and a better understanding of the mechanisms underlying the developmental plasticity of the eye and the development of neoplasms.

MHC Class I-Dependent Shaping of the NK Cell Ly49 Receptor Repertoire Takes Place Early during Maturation in the Bone Marrow.

MHC class I (MHC I) expression in the host influences NK cells in a process termed education. The result of this education is reflected in the responsiveness of NK cells at the level of individual cells as well as in the repertoire of inhibitory MHC I-specific receptors at the NK cell system level. The presence of MHC I molecules in the host environment gives rise to a skewed receptor repertoire in spleen NK cells where subsets expressing few (one or two) inhibitory receptors are expanded whereas subsets with many (three or more) receptors are contracted. It is not known whether this MHC I-dependent skewing is imposed during development or after maturation of NK cells. In this study, we tested the hypothesis that the NK cell receptor repertoire is shaped already early during NK cell development in the bone marrow. We used mice with a repertoire imposed by a single MHC I allele, as well as a C57BL/6 mutant strain with exaggerated repertoire skewing, to investigate Ly49 receptor repertoires at different stages of NK cell differentiation. Our results show that NK cell inhibitory receptor repertoire skewing can indeed be observed in the bone marrow, even during the earliest developmental steps where Ly49 receptors are expressed. This may partly be accounted for by selective proliferation of certain NK cell subsets, but other mechanisms must also be involved. We propose a model for how repertoire skewing is established during a developmental phase in the bone marrow, based on sequential receptor expression as well as selective proliferation.

ScAPAmod: Profiling Alternative Polyadenylation Modalities in Single Cells from Single-Cell RNA-Seq Data.

Alternative polyadenylation (APA) is a key layer of gene expression regulation, and APA choice is finely modulated in cells. Advances in single-cell RNA-seq (scRNA-seq) have provided unprecedented opportunities to study APA in cell populations. However, existing studies that investigated APA in single cells were either confined to a few cells or focused on profiling APA dynamics between cell types or identifying APA sites. The diversity and pattern of APA usages on a genomic scale in single cells remains unappreciated. Here, we proposed an analysis framework based on a Gaussian mixture model, scAPAmod, to identify patterns of APA usage from homogeneous or heterogeneous cell populations at the single-cell level. We systematically evaluated the performance of scAPAmod using simulated data and scRNA-seq data. The results show that scAPAmod can accurately identify different patterns of APA usages at the single-cell level. We analyzed the dynamic changes in the pattern of APA usage using scAPAmod in different cell differentiation and developmental stages during mouse spermatogenesis and found that even the same gene has different patterns of APA usages in different differentiation stages. The preference of patterns of usages of APA sites in different genomic regions was also analyzed. We found that patterns of APA usages of the same gene in 3′ UTRs (3′ untranslated region) and non-3′ UTRs are different. Moreover, we analyzed cell-type-specific APA usage patterns and changes in patterns of APA usages across cell types. Different from the conventional analysis of single-cell heterogeneity based on gene expression profiling, this study profiled the heterogeneous pattern of APA isoforms, which contributes to revealing the heterogeneity of single-cell gene expression with higher resolution.

LB1016 Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer

In the mammalian skin epidermis, it has long been unclear how molecularly heterogenous stem/progenitor cell populations fit into the complete trajectory of epidermal differentiation. Here, we combine in vivo imaging with single-cell RNA sequencing to define the early stages of epidermal stem cell differentiation at the molecular and behavioral level. We show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a gradual continuum of transcriptional changes. By tracking epidermal regions over many days, we find that differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions occur as a response to local density changes, and are not required as part of an obligate transit amplifying program but instead help to buffer the differentiating cell pool upon disruption of the epidermal barrier. Thus, instead of distinct contributions from multiple progenitor types, a single continuous differentiation process shaped by feedback from the local environment fuels lifelong turnover of the epidermis.

The human cathelicidin hCAP ‐18 in serum of children with haemato‐oncological diseases

SummaryThe human cathelicidin hCAP‐18 (pro‐LL‐37) is the pro‐protein of the antimicrobial peptide LL‐37. hCAP‐18 can be produced by many different cell types; bone marrow neutrophil precursors are the main source of hCAP‐18 in the circulation. Neutrophil count is used as a marker for myelopoiesis but does not always reflect neutrophil production in the bone marrow, and thus additional markers are needed. In this study, we established the reference interval of serum hCAP‐18 level in healthy children and compared serum hCAP‐18 levels between different diagnostic groups of children with haemato‐oncological diseases, at diagnosis. We found that children with diseases that impair myelopoiesis, such as acute leukaemia, aplastic anaemia, or myelodysplastic syndrome, presented with low hCAP‐18 levels, whereas patients with non‐haematological malignancies displayed serum hCAP‐18 levels in the same range as healthy children. Children with chronic myeloid leukaemia presented with high circulating levels of hCAP‐18, probably reflecting the high number of all differentiation stages of myeloid cells. We suggest that analysis of serum hCAP‐18 provides additional information regarding myelopoiesis in children with haemato‐oncological diseases, which may have future implications in assessment of myelopoiesis in clinical management.

M 6 Aexpress-BHM: predicting m6A regulation of gene expression in multiple-groups context by a Bayesian hierarchical mixture model.

As the most abundant RNA modification, N6-methyladenosine (m6A) plays an important role in various RNA activities including gene expression and translation. With the rapid application of MeRIP-seq technology, samples of multiple groups, such as the involved multiple viral/ bacterial infection or distinct cell differentiation stages, are extracted from same experimental unit. However, our current knowledge about how the dynamic m6A regulating gene expression and the role in certain biological processes (e.g. immune response in this complex context) is largely elusive due to lack of effective tools. To address this issue, we proposed a Bayesian hierarchical mixture model (called m6Aexpress-BHM) to predict m6A regulation of gene expression (m6A-reg-exp) in multiple groups of MeRIP-seq experiment with limited samples. Comprehensive evaluations of m6Aexpress-BHM on the simulated data demonstrate its high predicting precision and robustness. Applying m6Aexpress-BHM on three real-world datasets (i.e. Flaviviridae infection, infected time-points of bacteria and differentiation stages of dendritic cells), we predicted more m6A-reg-exp genes with positive regulatory mode that significantly participate in innate immune or adaptive immune pathways, revealing the underlying mechanism of the regulatory function of m6A during immune response. In addition, we also found that m6A may influence the expression of PD-1/PD-L1 via regulating its interacted genes. These results demonstrate the power of m6Aexpress-BHM, helping us understand the m6A regulatory function in immune system.

An immature, dedifferentiated, and lineage-deconstrained cone precursor origin of N-Myc–initiated retinoblastoma

Most retinoblastomas develop from maturing cone precursors in response to biallelic RB1 loss and are dependent on cone maturation-related signaling. Additionally, ∼2% lack RB1 mutations but have MYCN amplification (MYCNA), N-Myc protein overexpression, and more rapid and invasive growth, yet the MYCNA retinoblastoma cell of origin and basis for its responses to deregulated N-Myc are unknown. Here, using explanted cultured retinae, we show that ectopic N-Myc induces cell cycle entry in cells expressing markers of several retinal types yet induces continuous proliferation and tumorigenesis only in cone precursors. Unlike the response to RB1 loss, both immature cone arrestin-negative (ARR3-) and maturing ARR3+ cone precursors proliferate, and maturing cone precursors rapidly dedifferentiate, losing ARR3 as well as L/M-opsin expression. N-Myc-overexpressing retinal cells also lose cell lineage constraints, occasionally coexpressing the cone-specific RXRγ with the rod-specific NRL or amacrine-specific AP2α and widely coexpressing RXRγ with the progenitor and Müller cell-specific SOX9 and retinal ganglion cell-specific BRN3 and GAP43. Mechanistically, N-Myc induced Cyclin D2 and CDK4 overexpression, pRB phosphorylation, and SOX9-dependent proliferation without a retinoma-like stage that characterizes pRB-deficient retinoblastoma, despite continuous p16INK4A expression. Orthotopic xenografts of N-Myc-overexpressing retinal cells formed tumors with retinal cell marker expression similar to those in MYCN-transduced retinae and MYCNA retinoblastomas in patients. These findings demonstrate the MYCNA retinoblastoma origin from immature and lineage-deconstrained cone precursors, reveal their opportunistic use of an undifferentiated retinal progenitor cell feature, and illustrate that different cancer-initiating mutations cooperate with distinct developmental stage-specific cell signaling circuitries to drive retinoblastoma tumorigenesis.

Long Intergenic Noncoding RNA MIAT as a Regulator of Human Th17 Cell Differentiation.

T helper 17 (Th17) cells protect against fungal and bacterial infections and are implicated in autoimmunity. Several long intergenic noncoding RNAs (lincRNA) are induced during Th17 differentiation, however, their contribution to Th17 differentiation is poorly understood. We aimed to characterize the function of the lincRNA Myocardial Infarction Associated Transcript (MIAT) during early human Th17 cell differentiation. We found MIAT to be upregulated early after induction of human Th17 cell differentiation along with an increase in the chromatin accessibility at the gene locus. STAT3, a key regulator of Th17 differentiation, directly bound to the MIAT promoter and induced its expression during the early stages of Th17 cell differentiation. MIAT resides in the nucleus and regulates the expression of several key Th17 genes, including IL17A, IL17F, CCR6 and CXCL13, possibly by altering the chromatin accessibility of key loci, including IL17A locus. Further, MIAT regulates the expression of protein kinase C alpha (PKCα), an upstream regulator of IL17A. A reanalysis of published single-cell RNA-seq data showed that MIAT was expressed in T cells from the synovium of RA patients. Our results demonstrate that MIAT contributes to human Th17 differentiation by upregulating several genes implicated in Th17 differentiation. High MIAT expression in T cells of RA patient synovia suggests a possible role of MIAT in Th17 mediated autoimmune pathologies.

Esrrb Regulates Specific Feed-Forward Loops to Transit From Pluripotency Into Early Stages of Differentiation.

Characterization of pluripotent states, in which cells can both self-renew or differentiate, with the irreversible loss of pluripotency, are important research areas in developmental biology. Although microRNAs (miRNAs) have been shown to play a relevant role in cellular differentiation, the role of miRNAs integrated into gene regulatory networks and its dynamic changes during these early stages of embryonic stem cell (ESC) differentiation remain elusive. Here we describe the dynamic transcriptional regulatory circuitry of stem cells that incorporate protein-coding and miRNA genes based on miRNA array expression and quantitative sequencing of short transcripts upon the downregulation of the Estrogen Related Receptor Beta (Esrrb). The data reveals how Esrrb, a key stem cell transcription factor, regulates a specific stem cell miRNA expression program and integrates dynamic changes of feed-forward loops contributing to the early stages of cell differentiation upon its downregulation. Together these findings provide new insights on the architecture of the combined transcriptional post-transcriptional regulatory network in embryonic stem cells.

Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors

AbstractMast cell accumulation is a hallmark of a number of diseases, including allergic asthma and systemic mastocytosis. Immunoglobulin E–mediated crosslinking of the FcεRI receptors causes mast cell activation and contributes to disease pathogenesis. The mast cell lineage is one of the least studied among the hematopoietic cell lineages, and controversies remain about whether FcεRI expression appears during the mast cell progenitor stage or during terminal mast cell maturation. Here, we used single-cell transcriptomics analysis to reveal a temporal association between the appearance of FcεRI and the mast cell gene signature in CD34+ hematopoietic progenitors in adult peripheral blood. In agreement with these data, the FcεRI+ hematopoietic progenitors formed morphologically, phenotypically, and functionally mature mast cells in long-term culture assays. Single-cell transcriptomics analysis further revealed the expression patterns of prospective cytokine receptors regulating development of mast cell progenitors. Culture assays showed that interleukin-3 (IL-3) and IL-5 promoted disparate effects on progenitor cell proliferation and survival, respectively, whereas IL-33 caused robust FcεRI downregulation. Taken together, we showed that FcεRI expression appears at the progenitor stage of mast cell differentiation in peripheral blood. We also showed that external stimuli regulate FcεRI expression of mast cell progenitors, providing a possible explanation for the variable FcεRI expression levels during mast cell development.

Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation.

In cells, proteins encoded by the same gene do not all behave uniformly but engage in functional subpopulations induced by spatial or temporal segregation. While conventional microscopy has limitations in revealing such spatial and temporal diversity, single-molecule tracking (SMT) microscopy circumvented this problem and allows for high-resolution imaging and quantification of dynamic single-molecule properties. Particularly in the nucleus, SMT has identified specific DNA residence times of transcription factors (TFs), DNA-bound TF fractions and positions of transcriptional hot-spots upon cell stimulation. By contrast to cell stimulation, SMT has not been employed to follow dynamic TF changes along stages of cell differentiation. Herein, we analysed the serum response factor (SRF), a TF involved in the differentiation of many cell types to study nuclear single-molecule dynamics in neuronal differentiation. Our data in living mouse hippocampal neurons show dynamic changes in SRF DNA residence time and SRF DNA-bound fraction between the stages of adhesion, neurite growth and neurite differentiation in axon and dendrites. Using TALM (tracking and localization microscopy), we identified nuclear positions of SRF clusters and observed changes in their numbers and size during differentiation. Furthermore, we show that the SRF cofactor MRTF-A (myocardin-related TF or MKL1) responds to cell activation by enhancing the long-bound DNA fraction. Finally, a first SMT colocalization study of two proteins was performed in living cells showing enhanced SRF/MRTF-A colocalization upon stimulation. In summary, SMT revealed modulation of dynamic TF properties during cell stimulation and differentiation.

Glucocorticoids inhibit the maturation of committed osteoblasts via SOX2.

During bone formation, mesenchymal progenitor cells mature into bone-forming osteoblasts after undergoing several stages of differentiation. Impaired bone formation is a predominant finding in glucocorticoid (GC)-induced osteoporosis (GIO). Osteoblasts at different stages of maturation can be affected by excessive endogenous or therapeutic GCs. Sex-determining region Y-box 2 (SOX2) is normally expressed in immature osteoblasts, but its overexpression can suppress osteoblast differentiation. This study aimed to evaluate whether GC affects SOX2 expression in osteoblasts, and whether SOX2 contributes to GC-induced inhibition of osteoblast differentiation. Treatment with GCs such as dexamethasone (Dex) or hydrocortisone enhanced SOX2 expression. Silencing SOX2 improved inhibition of GC-induced osteoblast differentiation, whereas SOX2 overexpression decreased mineralized nodule formation and RUNX2 and Osterix expression in MC3T3-E1 cells. On the contrary, when C3H10T1/2 uncommitted mesenchymal stem cells were subjected to SOX2 overexpression, RUNX2 expression increased. As a mechanism of Dex-induced SOX2 upregulation in preosteoblasts, we found that the STAT3 pathway or GC receptor (GR) is involved, using a GR antagonist, STAT3 regulators, and chromatin immunoprecipitation assays. Moreover, mice treated with Dex for 4 weeks showed a notable increase in SOX2 expression in the bones and an increased ratio of procollagen type 1 N-terminal propeptide to osteocalcin in the plasma than in control mice. This study demonstrated that GC enhances SOX2 expression in vitro in osteoblast and in vivo in the mice bone, which affects bone-forming activity differently depending on the differentiation stage of osteoblast-lineage cells. Our results provide new insights into prevention and treatment against impaired bone formation in GIO.

Advanced Immune Cell Profiling by Multiparameter Flow Cytometry in Humanized Patient-Derived Tumor Mice.

Simple SummaryImmunotherapies have revolutionized the field of oncology and have been approved to treat cancer. Despite progress in immunotherapy, many challenges remain, including the identification (i) of predictive markers for treatment response or (ii) of beneficial T cell subsets involved in tumor elimination. “Humanized” mice are a promising translational model for studying the human immune system in the context of immuno-oncology research. Here, multicolor flow cytometry was applied to characterize immune cell subsets in the spleen of humanized mice transplanted with patient-derived breast cancer tissues. This multicolor immune cell setup will help to identify promising therapeutic approaches or predictive immune cell subsets in the future using humanized tumor mice.“Humanized” mice have been widely used for the characterization of human cancer progression and as a powerful preclinical model. Standardization of multicolor phenotyping could help to identify immune cell patterns involved in checkpoint-related complications. Therefore, we applied established protocols for immune cell profiling to our humanized Patient-Derived Xenograft (hPDX) model. hPDX are characterized by the co-existence of a human immune system and a patient-derived tumor transplant. These mice possess a human-like immune system after CD34+ stem cell transplantation while the reconstitution level of the immune system was not related to the quantity of transplanted CD34+ cells. Contamination ≤ 1.2% by CD3+ cells in the hematopoietic stem cell (HSC) transplant did not trigger abnormal T cell maturation. Different B and T cell differentiation stages were identified, as well as regulatory T cells (Tregs) and exhausted T cells that expressed TIGIT, PD-1, or KLRG1. Overall, the application of standardized protocols for the characterization of immune cells using flow cytometry will contribute to a better understanding of immune-oncologic processes.

Ferromagnetic Vortex Iron Oxide Nanorings Modified with Integrin β1 Antibody for Targeted MRI Tracking of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have demonstrated great potential for tissue engineering and regenerative medicine applications. Noninvasive and real-term tracking of transplanted MSCs in vivo is crucial for studying the distribution and migration of MSCs, and their role in tissue injury repair. This study reports on the use of ferrimagnetic vortex iron oxide (FVIO) nanorings modified with anti-human integrin β1 for specific recognition and magnetic resonance imaging (MRI) tracking of human MSCs (hMSCs). Integrin β1 is highly expressed at all stem cell proliferation and differentiation stages. Therefore, the anti-integrin β1 antibody (Ab) introduced in FVIO targets integrin β1, thus enabling FVIO to target stem cells at any stage. This is unlike the traditional MRI-based monitoring of transplanted stem cells, which usually requires pre-labeling the stem cells with tracers before injection. Because of the ability to recognize hMSCs, the Ab-modified FVIO nanotracers (FVIO-Ab) have the advantage of not requiring pre-labeling before stem cell transplantation. Furthermore, the FVIO-Ab nanotracers have high T*₂ contrast resulting from the unique magnetic properties of FVIO which can improve the MRI tracking efficiency of stem cells. This work may provide a new way for stem cell labeling and in vivo MRI tracking, thus reducing the risks associated with stem cell transplantation and promoting clinical translation.

Characterisation of induced Malus x domestica ‘Royal Gala’ cell differentiation by using different hormones in cell cultures

ABSTRACT Fruit development, following pollination, involves a cell division stage, followed by cell expansion . A major challenges to study each individual stages is the limitations imposed by analyses of fruit development on the plant. Rapid growing cell culture systems that differentiate could be used to identify and understand the molecular control of each of these processes in vitro. In this study, an apple cell culture was initiated from the widely characterised ‘Royal Gala’ cultivar. The established apple cell culture was used to develop a screening system to assess the effect of different hormones on cell differentiation, which then were characterised at molecular and cellular levels. Three different hormone pairs were screened to determine the optimal for cell expansion on actively dividing cells . Using this strategy, we were able to reproduce, in vitro, the first two stages of apple fruit cell differentiation, cell division and expansion, which was correlated with changes in genes expression of specific markers for those two stages, a cyclin-dependent kinase gene (CDKB2;2) and an expansion gene (EXP3). The cell culture provides a unique tool and opportunity to study the effect of plant hormones on cell differentiation and will allow to decipher the molecular mechanism of apple fruit development irrespective of y.

Quantitative karyological analysis of immature germ cells from ejaculate with normal sperm count

Introduction. The number of immature germ cells (IGCs) in ejaculate and the ratio of these cells at different stages of their differentiation can reflect meiotic progression and the state of spermatogenesis. Quantitative karyological analysis of immature germ cells from the ejaculate (QKA IGCs) makes it possible to distinguish and analyze all the stages of cell differentiation during spermatogenesis.Purpose. To analyze the spermatogenesis in men with impaired fertility and possible correlation between semen analysis and QKA IGCs parameters. Materials and methods. We analyzed 52 semen samples with normal sperm concentration, normal ejaculate volume and pH. Sperm examination according to WHO criteria and QKA IGCs from the ejaculate were performed (patent of L.F. Kurilo No. 2328736, 2007). According to the number of leukocytes, we divided two groups: a group with normal concentration of leukocytes in the ejaculate (group 1, n = 34) and a group with leukospermia (group 2, n = 18).Results. Increased IGCs index was found in 56 % of the samples in group 1 and 61 % of the samples in group 2. A positive statistically significant correlation was found between the number of spermatocytes I and sperm concentration (group 1), as well as the number of spermatocytes I and morphologically normal spermatozoa (group 2). The average number of non-divided spermatids in group 2 was statistically higher than in group 1. A negative statistically significant correlation was found between the number of non-divided spermatids and the number of morphologically normal spermatozoa, as well as the concentration of germ cells (group 2). Increased IGCs index was found in both pathozoospermia and normozoospermia.Conclusions. The QKA IGCs is an independent method for spermatogenesis evaluation and can be recommended as part of the protocol for examining men with reproductive disorders along with standard semen analysis.

Two independent cis-acting elements are required for the guard cell-specific expression of SCAP1, which is essential for late stomatal development.

Regulating the stomatal aperture to adapt to environmental changes is critical for plants as stomatal guard cells are responsible for gas exchange between plants and the atmosphere. We previously showed that a plant-specific DNA-binding with one finger (Dof)-type transcription factor, SCAP1, functions as a key regulator in the final stages of guard cell differentiation. In the present study, we performed deletion and gain-of-function analyses with the 5' flanking region of SCAP1 to identify the regulatory region controlling the guard cell-specific expression of SCAP1. The results revealed that two cis-acting elements, 5'-CACGAGA-3' and 5'-CACATGTTTCCC-3', are crucial for the guard cell-specific expression of SCAP1. Consistently, when an 80-bp promoter region including these two cis-elements was fused to a gene promoter that is not active in guard cells, it functioned as a promoter that directed gene expression in guard cells. Furthermore, the promoter region of HT1 encoding the central regulator of stomatal CO2 signaling was also found to contain a 5'-CACGAGA-3' sequence, which was confirmed to function as a cis-element necessary for guard cell-specific expression of HT1. These findings suggest the existence of a novel transcriptional regulatory mechanism that synchronously promotes the expression of multiple genes required for the stomatal maturation and function.

Norisoboldine induces the development of Treg cells by promoting fatty acid oxidation-mediated H3K27 acetylation of Foxp3.

Norisoboldine (NOR), an alkaloid isolated from Radix Lindera, was previously reported to promote the differentiation of regulatory T cells (Treg cells), an important subtype of lymphocytes capable of controlling autoimmune diseases. The present study was performed to explore the mechanism of NOR in the view of cellular metabolism. A global metabolomic analysis indicated that NOR preferentially altered the fatty acid oxidation (FAO) pathway and elevated the content of related metabolites during Treg cell differentiation. The detection of oxygen consumption rate (OCR) and mRNA expression of FAO-related enzymes demonstrated that NOR promoted FAO in the early stage of Treg cell differentiation. Consistently, pharmacological or genetic inhibition of FAO markedly diminished the induction of NOR on Treg cell differentiation. Furthermore, NOR was shown to elevate the level of acetyl-CoA derived from FAO and acetylation of lysine 27 on histone 3 (H3K27) at the Foxp3 promoter and CNS2 regions. A knockdown of CPT1, the rate-limiting enzyme of FAO, weakened the promotion of NOR on the development, acetyl-CoA level, and acetylation of H3K27 of Treg cells in vitro and in the mice with collagen-induced arthritis, and attenuated the anti-arthritic effect of NOR. These findings demonstrate that NOR induces the development of Treg cells through promoting FAO, therefore, facilitating gene transcription of Foxp3 via acetyl-CoA-mediated H3K27 acetylation modification, and FAO might serve as a novel target to induce Treg cell development.