Chemical modulation of cell fates: <Emphasis Type="Italic">in situ</Emphasis> regeneration

Chemical modulation of cell fates has been widely used to promote tissue and organ regeneration. Small molecules can target the self-renewal, expansion, differentiation, and survival of endogenous stem cells for enhancing their regenerative power or induce dedifferentiation or transdifferentiation of mature cells into proliferative progenitors or specialized cell types needed for regeneration. Here, we discuss current progress and potential using small molecules to promote in vivo regenerative processes by regulating the cell fate. Current studies of small molecules in regeneration will provide insights into developing safe and efficient chemical approaches for in situ tissue repair and regeneration.     

A functional genomics screen identifies an Importin-α homolog as a regulator of stem cell function and tissue patterning during planarian regeneration

Background

Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea.

Methods

We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians regenerating one side of the head and followed this with high-throughput screening by in situ hybridization and RNAi to characterize the expression patterns and function of the differentially expressed genes.

Results

Along with five previously characterized genes (Smed-cycD, Smed-morf41/mrg-1, Smed-pdss2/dlp1, Smed-slbp, and Smed-tph), we identified 20 additional genes necessary for stem cell maintenance (Smed-sart3, Smed-smarcc-1, Smed-espl1, Smed-rrm2b-1, Smed-rrm2b-2, Smed-dkc1, Smed-emg1, Smed-lig1, Smed-prim2, Smed-mcm7, and a novel sequence) or general regenerative capability (Smed-rbap46/48-2, Smed-mcm2, Smed-ptbp1, and Smed-fen-1) or that caused tissue-specific defects upon knockdown (Smed-ddc, Smed-gas8, Smed-pgbd4, and Smed-b9d2). We also found that a homolog of the nuclear transport factor Importin-α plays a role in stem cell function and tissue patterning, suggesting that controlled nuclear import of proteins is important for regeneration.

Conclusions

Through this work, we described the roles of several previously uncharacterized genes in planarian regeneration and implicated nuclear import in this process. We have additionally created an online database to house our in situ and RNAi data to make it accessible to the planarian research community.

     

Impact of early subcultures on stemness,migration and angiogenic potential of adipose tissue-derived stem cells and their resistance to in vitro ischemic condition

Adipose tissue-derived stem cells (ADSCs) are capable of multipotential differentiation and express several angiogenic, anti-apoptotic and immunomodulatory markers. These features make adipose tissue as a promising source of stem cells for regenerative medicine. However, for efficient translational use, culture-induced changes in the gene expression profile and resistance of the ADSCs to ischemic environment should be taken into consideration. We compared the expression of some clinically important markers between the unpassaged and third-passaged ADSCs by RT-PCR, qPCR and flow cytometry. Our results demonstrated that the embryonic stem cell (ESC)-specific markers were expressed in the unpassaged ADSCs but were downregulated after three passages. The expression of stemness-related genes, TGFB and FGF2, was upregulated while FGF4 and LIF were downregulated after three passages. The expression of angiogenic genes in the third-passaged ADSCs was higher than the unpassaged cells. Epithelial-mesenchymal transition (EMT) markers were either expressed in the third-passaged ADSCs or significantly upregulated after three passages. In contrast, cell cycle inhibitors, CDKN1A and TP53, were downregulated with early subcultures. The unpassaged and third-passaged ADSCs showed nearly similar resistance to oxidative stress, hypoxia and serum deprivation. In conclusion, the primary cultures of human adipose tissue contain a subpopulation of cells expressing ESC-specific genes and proteins, but the expression of these pluripotency markers subsides rapidly in standard mesenchymal stem cell culture medium. The expression of angiogenic and EMT markers also varies with early subcultures. Altogether, early-passaged ADSCs may be better choices for transplantation therapy of injured tissues, especially after ischemic conditions.     

Anatomical and micromorphological studies on <Emphasis Type="Italic">Teucrium</Emphasis> sect. <Emphasis Type="Italic">Isotriodon</Emphasis> (Lamiaceae) in Turkey with a taxonomic note

In this study, the anatomical features of the leaf and stem, besides the nutlet characteristics of some Teucrium sect. Isotriodon (Lamiaceae) taxa in Turkey, T. montbretii Betham subsp. montbretii, T. montbretii subsp. pamphylicum P. H. Davis, T. odontites Boiss. &; Bal., T. cavernarum P. H. Davis, T. antitauricum T. Ekim, along with an isolated population of T. montbretii (T. montbretii subsp.) were investigated. The anatomical studies revealed that the taxa share generally similar anatomical characters, such as thicker upper leaf cuticles and larger upper leaf epidermal cells compared to lower ones and diacytic to anomocytic stomata on the leaves. However, the portion of the mesophyll occupied by palisade parenchyma and the occurrence of mucilage cells in leaf epidermis shows difference among the taxa. Furthermore, the studied taxa have general stem characteristics of the Lamiaceae family, except for having poorly developed collenchyma at the corners. With the amphistomatic leaves and developed sclerenchymatic tissue in the leaf median vein, T. cavernarum is seperated from the other taxa. Trichome types on the vegetative organs and nutlet shape and sculpturing are generally the same or similar in the studied taxa, but trichomes on the nutlets are different among them. Based on nutlet characteristics and some morphological ones, it was revealed that the isolated population of T. montbretii represent a new subspecies, T. monbretii subsp. yildirimlii M.Dinç &; S.Do?u subsp. nov.     

Generation of an <Emphasis Type="Italic">Rtel1</Emphasis>-<Emphasis Type="Italic">CreERT2</Emphasis> knock-in mouse model for lineage tracing RTEL1+ stem cells during development

Regulator of telomere length 1 (RTEL1) DNA helicase has been demonstrated to be essential for the maintenance of telomeres and genomic stability. This function of RTEL1 could be required for protecting stem cells from genomic mutations as suggested by its selective expression in stem cell-zones, as well as by RTEL1 mutations identified in Hoyeraal-Hreidarsson syndrome, a severe dyskeratosis congenita that targets primarily stem cell compartments. As a first step to establish a role of RTEL1 in stem cells, we generated an Rtel1^CreERT2 mouse allele in which a tamoxifen-inducible Cre (CreERT2) cDNA was specifically knocked into the Rtel1 genomic locus and controlled by the endogenous Rtel1 regulatory elements. By crossing with a Cre-dependent LacZ reporter mouse strain (R26R^LacZ), we further demonstrated that Cre activity in Rtel1^CreERT2 mice could be specifically induced by tamoxifen, which allowed the fate of RTEL1+ cells to be traced at various stages of development. Using this tracing assay, we showed for the first time that RTEL1+ cells in the intestine and the testis can act as stem cells that have the capacity to self-renew and differentiate into progeny cells. Therefore, the Rtel1^CreERT2 mice generated in this study will be a valuable transgenic tool to explore the function of RTEL1 in stem cells.     

Structural and quantitative evidence of α2–6-sialylated <Emphasis Type="Italic">N</Emphasis>-glycans as markers of the differentiation potential of human mesenchymal stem cells

Human somatic stem cells such as mesenchymal stem cells (hMSCs) have the capacity to differentiate into mesenchymal tissue lineages and to alter immune regulatory functions. As such, they hold promise for use in stem cell-based therapies. However, no method is currently available to evaluate the actual differentiation capacity of hMSCs prior to cell transplantation. Previously, we performed a comprehensive glycan profiling of adipose-derived hMSCs using high-density lectin microarray and demonstrated that α2–6-sialylation is a marker of the differentiation potential of these cells. Nevertheless, no information was available about the structural details of these of α2–6-sialylated glycans. Here we used high performance liquid chromatography (HPLC) analysis combined with mass spectrometry (MS) to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from early (with differentiation ability) and late (without differentiation ability) passages of adipose tissue-derived hMSCs. Findings in these cells were compared with those from human induced pluripotent stem cells (hiPSCs), human dermal fibroblasts (hFibs) and cartilage tissue-derived chondrocytes. A higher percentage of α2–6-sialylated N-glycans was detected in early passage cells (24–28 % of sialylated N-glycans) compared with late passage cells (13–15 %). A major α2–6-sialylated N-glycan structure detected in adipose-derived hMSCs was that of mono-sialylated biantennary N-glycan. Similar results were obtained for the cartilage tissue-derived chondrocytes, Yub621c (28 % for passage 7 and 5 % for passage 28). In contrast, no significant differences were observed between early and late passage hMSCs with respect to α2–6-sialylated O-glycan percentages. These results demonstrate that levels of α2–6-sialylated N-glycans, but not O-glycans, could be used as markers of the differential potential of hMSCs.     

Interactions between bradykinin and plasmin in the endothelial Ca<Superscript>2+</Superscript> response

Frequent abnormalities in 7p12 locus in different tumors like lung cancer candidate this region for novel regulatory elements. MiRNAs as novel regulatory elements encoded within the human genome are potentially oncomiRs or miR suppressors. Here, we have used bioinformatics tools to search for the novel miRNAs embedded within human chromosome 7p12. A bona fide stem loop (named mirZa precursor) had the features of producing a real miRNA (named miRZa) which was detected through RT-qPCR following the overexpression of its precursor. Then, endogenous miRZa was detected in human cell lines and tissues and sequenced. Consistent to the bioinformatics prediction, RT-qPCR as well as dual luciferase assay indicated that SMAD3 and IGF1R genes were targeted by miRZa. MiRZa-3p and miRZa-5p were downregulated in lung tumor tissue samples detected by RT-qPCR, and mirZa precursor overexpression in SW480 cells resulted in increased sub-G1 cell population. Overall, here we introduced a novel miRNA which is capable of targeting SMAD3 and IGF1R regulatory genes and increases the cell population in sub-G1 stage.     

A role for the endometrial microbiome in dysfunctional menstrual bleeding

This study aimed to characterise the microbial community within the endometrial cavity and endocervix in women with menorrhagia or dysmenorrhea. Paired endocervical and endometrial biopsy samples were collected from women undergoing operative hysteroscopy and/or laparoscopy. Samples were cohorted based on pathology, indications for surgery, and histological dating of the endometrium. Samples were interrogated for the presence of microbial DNA using a two-step next generation sequencing technology approach to exploit the V5–V8 regions of the 16S rRNA gene. Pyrosequencing revealed that the endocervix and endometrium share a minor microbial community, but that each site harbours a separate and distinct microbial population (p = 0.024). This was also the case for women with menorrhagia and dysmenorrhea (p = 0.017). Lactobacillus spp. were the most abundant microbial taxa present in 50% of the cohorts, and across all endocervical groups. Members of the genera Prevotella, Fusobacterium and Jonquetella were the most abundant taxa identified in samples collected from nulliparous women. It can be concluded that the female upper genital tract is not sterile. Microbial community profiling revealed differences in the endometrial microbial community profiles for: (1) the endocervix compared to the endometrium, and (2), women with menorrhagia versus dysmenorrhea. The distinct microbial community profiles in these women may offer insight into the pathology and clinical management of dysfunctional menstrual bleeding.     

Morphofunctional organization of reserve stem cells providing for asexual and sexual reproduction of invertebrates

Published and original data indicating evolutionary conservation of the morphofunctional organization of reserve stem cells providing for asexual and sexual reproduction of invertebrates are reviewed. Stem cells were studied in representatives of five animal types: archeocytes in sponge Oscarella malakhovi (Porifera), large interstitial cells in colonial hydroid Obelia longissima (Cnidaria), neoblasts in an asexual race of planarian Girardia tigrina (Platyhelmintes), stem cells in colonial rhizocephalans Peltogasterella gracilis, Polyascus polygenea, and Thylacoplethus isaevae (Arthropoda), and colonial ascidian Botryllus tuberatus (Chordata). Stem cells in animals of such diverse taxa feature the presence of germinal granules, are positive for proliferating cell nuclear antigen, demonstrate alkaline phosphatase activity (a marker of embryonic stem cells and primary germ cells in vertebrates), and rhizocephalan stem cells express the vasa-like gene (such genes are expressed in germline cells of different metazoans). The self-renewing pool of stem cells is the cellular basis of the reproductive strategy including sexual and asexual reproduction.     

Engineered Co-culture Strategies Using Stem Cells for Facilitated Chondrogenic Differentiation and Cartilage Repair

Osteoarthritis (OA) is a chronic disease in elders and athletes due to limited regenerative capacities of cartilage tissues and subsequently insufficient recovery of damaged sites. Recent clinical treatments for OA have utilized progenitor cell-based therapies for cartilage tissue regeneration. Administration of a single type of cell population such as stem cells or chondrocytes does not guarantee a full recovery of cartilage defects. Therefore, current tissue engineering approaches using co-culture techniques have been developed to mimic complex and dynamic cellular interactions in native cartilage tissues and facilitate changes in cellular phenotypes into chondrogenesis. Therefore, this paper introduces recently developed co-culture systems using two major cell populations, mesenchymal stem cells (MSCs) and chondrocytes. Specifically, a series of examples to describe (1) synergistic in vitro activations of MSCs by paracrine signaling molecules from adult chondrocytes in co-culture systems and (2) functional in vivo tissue regeneration via co-administration of both cell types were reviewed. Based on these findings, it could be speculated that engineered co-culture systems using MSC/ chondrocyte is a promising and feasible cell-based OA therapy in clinical aspects.     

Mathematical models for cellular systems. The von foerster equation. Part II

This is the continuation of Part I, which was published in the September, 1965, issue of theBulletin. The birth rate, α(t), is now assumed to be a linear functional of the age density,n. This gives a simple model of self-replenishing stem cell compartments, and leads to a necessary condition for the existence of a steady state. Some examples are presented to illustrate the formalism. They include: (a) An equivivant population with life spanD and no losses from death or migration. The total number of cells is multiplied by 2 in each time intervalD. As a special case, frequently realized in practice, the population may be increasing exponentially with time (“log-phase” of growth). (b) A compartment with “random” emigration of cells and gamma distribution of life spans. (c) An oversimplified version of L. G. Lajtha’s model describing stem cell kinetics. In section IV a simple case in which the loss function depends explicitly onn is discussed very briefly.     

Boric Acid Inhibition of <Emphasis Type="Italic">Trichophyton rubrum</Emphasis> Growth and Conidia Formation

Trichophyton rubrum is a common human dermatophyte that is the causative agent of 80–93% of fungal infections of the skin and nails. While dermatophyte infections in healthy people are easily treatable with over-the-counter medications, such infections pose a higher risk for patients with compromised immune function and impaired regenerative potential. The efficacy of boric acid (BA) for the treatment of vaginal yeast infections prompted an investigation of the effect of BA on growth and morphology of T. rubrum. This is of particular interest since BA facilitates wound healing, raising the possibility that treating athlete’s foot with BA, either alone or in combination with other antifungal drugs, would combine the benefits of antimicrobial activity and tissue regeneration to accelerate healing of infected skin. The data presented here show that BA represses T. rubrum growth at a concentration reported to be beneficial for host tissue regeneration. Oxygen exposure increases BA toxicity, and mycelia growing under BA stress avoid colonizing the surface of the growth surface, which leads to a suppression of aerial mycelium growth and surface conidia formation. BA penetrates into solid agar matrices, but the relative lack of oxygen below the substrate surface limits the effectiveness of BA in suppressing growth of embedded T. rubrum cells.     

The Differentiation of Skin Mesenchymal Stem Cells Towards a Schwann Cell Phenotype: Impact of Sigma-1 Receptor Activation

Neural crest stem cells (NCSCs) are the source of mature Schwann cells in the peripheral nervous system (PNS). The NCSC population resides in the bulge of hair follicles and in the dermis. Recently, it was shown that 2–3% of the human dermis mesenchymal stem cell (MSC) population expresses the NCSC marker CD271, thus enabling the use of skin MSCs for studying Schwann cell differentiation in vitro. The aims of this study were to establish a protocol for human skin MSC differentiation towards Schwann cell-like cells (SC-lcs) and to analyse the expression of sigma-1 receptor (S1R) in SC-lcs. The impact of S1R ligands, namely the selective agonist PRE-084, the positive allosteric modulator E1R and the selective antagonist NE-100, on Schwann cell differentiation was assessed. The expression of the neuron-specific genes Tubulin-βIII and Integrin-6α, the Schwann cell-specific gene S100b, MBP and the NCSC-specific genes p75NTR, Sox10, Notch1, Integrin-4α, Ap2α and Pax6 was analysed in MSCs and SC-lcs by real-time RT-PCR. BDNF secretion was evaluated by ELISA. The effect of S1R ligands on SC-lc differentiation was measured using BDNF ELISA and MBP flow cytometry. After MSC differentiation, NCSC markers p75NTR and Integrin-4α were downregulated 3.5-fold and 2-fold, respectively. To the contrary, MBP and S100b were significantly upregulated in SC-lcs. S1R ligands showed a tendency to increase the secretion of BDNF by the SC-lc population. PRE-084 and E1R increased MBP expression in the SC-lc population, whereas 3 μM NE-100 inhibited MBP expression in SC-lcs. In conclusion, our data demonstrate that S1R plays an important role in skin MSC differentiation towards myelinating Schwann cells.     

Development of co-dominant KASP markers co-segregating with Ug99 effective stem rust resistance gene <Emphasis Type="Italic">Sr26</Emphasis> in wheat

Stem rust of wheat, caused by Puccinia graminis f. sp. tritici (Pgt), is a threat to global food security due to its ability to cause total crop failures. The Pgt race TTKSK (Ug99) and its derivatives detected in East Africa carry virulence for many resistance genes present in modern cultivars. However, stem rust resistance gene Sr26 remains effective to all races of Pgt worldwide. Sr26 is carried on the Agropyron elongatum (syn. Thinopyrum ponticum) segment 6Ae#1L translocated to chromosome 6AL of wheat. In this study, a recombinant inbred line (RIL) population derived from a cross between the landrace Aus27969 and Avocet S, which carries Sr26, was used to develop co-dominant kompetitive allele-specific polymerase chain reaction (KASP) markers that co-segregate with Sr26. Four KASP markers (sunKASP_216, sunKASP_218, sunKASP_224 and sunKASP_225) were also shown to co-segregate with Sr26 in four additional RIL populations. When tested on Australian cultivars and breeding lines, these markers amplified alleles alternate to that linked with Sr26 in all cultivars known to lack this gene and Sr26-linked alleles in cultivars and genotypes known to carry Sr26. Genotypes WA-1 and WA-1/3*Yitpi carrying the shortest Sr26 translocation segment were positive only for markers sunKASP_224 and sunKASP_225. Our results suggest the four KASP markers are located on the original translocation and sunKASP_224 and sunKASP_225 are located on the shortened version. Therefore, sunKASP_224 and sunKASP_225 can be used for marker-assisted pyramiding of Sr26 with other stem rust resistance genes to achieve durable resistance in wheat.     

New species of Telenominae (Hymenoptera,Scelionidae) from the palaearctic fauna

Four new species of Telenominae of the genera Telenomus and Platytelenomus, collected in the territory of the Ukraine, Hungary, and Japan, were described: Telenomus (T.) bicolorus Kononova, T. (T.) ardens Kononova, (T.) michaylovi Kononova, and Platytelenomus mirabilis Kononova. Brief morphological characteristics of the genera Telenomus and Platytelenomus are given, and some notes concerning biology and geographical distribution of the species are presented. Telenomus (T.) bicolorus differs from all the known species of the genus Telenomus in the two-colored body: head and thorax yellow with brownish tint ventrally, mesothorax and abdomen black. The main distinguishing feature of T. (T.) ardens is its smooth shining body, T. (T.) michaylovi is similar to T. (T.) rudis Kozlov. These species can be distinguished by the structure of their antenna. The second to fourth segments of the antennal clava are transverse in T. (T.) michaylovi and are as long as wide in T. (T.) rudis. In addition, the abdominal stem and abdominal tergite II are smooth and shining, while the abdominal stem in T. (T.) rudis is striate along the entire length, and tergite II is finely striate along half of its length. Platytelenomus mirabilis is closely related to P. danubialis Szelényi, but differs in the strongly flattened body, sculpture of the abdominal stem and tergite II, and coloration of the legs. The thorax of P. mirabilis is 4–5 times as wide as high, the abdominal stem is striate along the entire length, tergite II is striate at the base, and the legs, including coxae, are yellow. The thorax of P. danubialis is 4 times as wide as high, the abdominal stem and tergite II are smooth and shining, and the legs are brown.     

Discovery and characterization of two new stem rust resistance genes in <Emphasis Type="Italic">Aegilops sharonensis</Emphasis>

Key message

We identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen.

Abstract

Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F₆ recombinant inbred line and an F₂ population, two genes were identified that mapped to the short arm of chromosome 1S^sh, designated as Sr-1644-1Sh, and the long arm of chromosome 5S^sh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.