Characterization and Functional Analysis of a Murine Model for Chronic Myeloid Leukemia Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4476-4476
Author(s):  
Selcuk Sozer ◽  
Debbie R. Echlin ◽  
Randall M. Rossi ◽  
Barry A. Grimes ◽  
Craig T. Jordan
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Leukemic Cells ◽  
Brdu Incorporation ◽  
Myeloproliferative Disease ◽  
Phenotypic Analysis ◽  
Potential Marker ◽  
Heat Stable Antigen ◽  
Green Fluorescent

Abstract The purpose of this study was to characterize the phenotype and analyze the functional characteristics of CML stem cells (CML-SCs) in a mouse model system. A retrovirus transduction model was employed using a vector expressing p210/BCR/ABL and the green fluorescent protein (GFP). Transplantation studies of BCR/ABL infected cells in recipient mice demonstrated an aggressive myeloproliferative disease within approximately 3 weeks, as has previously been observed with this approach. Phenotypic analysis of bone marrow from BCR/ABL mice showed contribution by the transduced cells to multiple lineages, suggesting a primitive stem/progenitor cell gives rise to the disease. Subsequent analysis of primary cells that arise in vivo showed the leukemia-initiating activity resides in cells that are Sca-1+/c-kit+/Lin-. Cell cycle analyses (as detected by BrdU incorporation) of the Lin−/c-kit+/Sca-1+ compartment of BCR/ABL mice demonstrated an enhanced cycle rate (approximately 2-fold) in the leukemic population, signifying a possible increase in the self-renewal or proliferative properties of this primitive compartment that could lead to development/progression of the disease. Further, comparison of the BCR/ABL mice and GFP control mice revealed preferential over-expression of CD24 (heat-stable antigen) in the primitive leukemic cells (90% vs. 52% in normal counterparts) demonstrating a possible BCR/ABL induced activation of the antigen. Sorting and serial transplantation studies of CD24+ vs CD24− cells from BCR/ABL mice showed that the leukemia-initiating activity resides in the CD24+/Lin− population. Collectively, these data indicate that BCR/ABL expression increases proliferation of primitive CML cells and up-regulates the CD24 antigen. We suggest that the CD24 antigen represents a potential marker to distinguish normal from CML stem cells and that it may confer specific functional properties on the CML population.

scholarly journals Hematopoietic stem cells can differentiate into pericytes and myofibroblast in wound healing, not bone Mesenchymal stem cells

2020 ◽  
Author(s):  
Yanan Kong ◽  
Liuhanghang Cheng ◽  
Min Xuan ◽  
Hao Ding ◽  
Biao Cheng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Wound Healing ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Bone Mesenchymal Stem Cells ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Background Hematopoietic stem cells(HSCs) and mesenchymal stem cells(MSCs) can participate in wound healing. However, very few studies had shown HSCs and MSCs could arrive to the wound and differentiate into tissues. In this study, we intend to investigate the role of bone marrow HSCs and MSCs in wound healing. Methods We first removed the bone marrow of mice by irradiation. Furthermore, we injected different colours of fluorescent HSCs and MSCs into the tail vein of irradiated mice to reconstruct bone marrow function. We prepared wound models on the back of these mice. In vivo imaging and immunohistochemical staining were used to track the expression of fluorescent protein. Results HSCs and MSCs have been isolated and cultured. HSCs expressed expressed Sca1, not lineage, CD34 or CD48. MSCs expressed expressed CD29 and CD44,not CD34 or CD45. HSCs labeled with green fluorescent protein reached the wound and co-expressed with desmin and α-SMA. MSCs didn’t stay on the wound. Conclusions The results show HSCs in the bone marrow of mice can directly participate in wound healing and differentiate into pericytes and myofibroblasts.

Effect of MicroRNA-20a on Osteogenic Differentiation of Human Adipose Tissue-Derived Stem Cells

2019 ◽  
Vol 208 (3-4) ◽  
pp. 148-157
Author(s):  
Tao Luo ◽  
Xueqin Yang ◽  
Yan Sun ◽  
Xinqi Huang ◽  
Ling Zou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Osteogenic Differentiation ◽  
Fluorescent Protein ◽  
Mrna Level ◽  
Human Adipose Tissue ◽  
Associated Proteins ◽  
Green Fluorescent

Osteogenic differentiation of human adipose tissue-derived stem cells (hASCs) is a complex process that is regulated by multiple factors, including microRNAs (miRNAs). The miRNA miR-20a was shown to promote bone formation from bone marrow-derived mesenchymal stem cells. However, the role of miR-20a in osteogenic differentiation of hASCs remains unclear. In this study, we systematically evaluated the function of miR-20a in regulating hASC osteogenesis in vitro. hASCs were transduced with miR-20a-overexpressing and miR-20a-sponge lentiviral vectors, with green fluorescent protein (GFP) as a control. The results showed that miR-20a transcription was upregulated after hASC mineralization. Compared with the miR-20a-sponge, GFP, and hASC groups, the miR-20a-overexpressing group showed higher alkaline phosphatase (ALP) activity on days 7 and 14. Moreover, the mRNA level of ALP increased significantly in the miR-20a-overexpressing group on day 14. Furthermore, the protein of the target gene PPARγ was decreased, and the osteogenic differentiation-associated proteins ALP, osteocalcin, and RUNX2 were upregulated. hASCs anchored to HA/β-TCP revealed a healthy polygonal morphology and developed cytoplasmic extensions. miR-20a promoted osteogenic differentiation of the cell scaffold. Taken together, these data ­confirm that miRNA-20a promotes the osteogenesis of hASCs in vitro, and its essential role in vivo needs further ­investigation.

scholarly journals The Anaerobic Product Ethanol Promotes Autophagy-Dependent Submergence Tolerance in Arabidopsis

2020 ◽  
Vol 21 (19) ◽  
pp. 7361 ◽  
Author(s):  
Li-Bing Yuan ◽  
Liang Chen ◽  
Ning Zhai ◽  
Ying Zhou ◽  
Shan-Shan Zhao ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Anaerobic Fermentation ◽  
Submergence Tolerance ◽  
Ethanol Treatment ◽  
Autophagosome Formation ◽  
Phenotypic Analysis ◽  
Atg Proteins ◽  
Transgenic Lines ◽  
Green Fluorescent

In response to hypoxia under submergence, plants switch from aerobic respiration to anaerobic fermentation, which leads to the accumulation of the end product, ethanol. We previously reported that Arabidopsis thaliana autophagy-deficient mutants show increased sensitivity to ethanol treatment, indicating that ethanol is likely involved in regulating the autophagy-mediated hypoxia response. Here, using a transcriptomic analysis, we identified 3909 genes in Arabidopsis seedlings that were differentially expressed in response to ethanol treatment, including 2487 upregulated and 1422 downregulated genes. Ethanol treatment significantly upregulated genes involved in autophagy and the detoxification of reactive oxygen species. Using transgenic lines expressing AUTOPHAGY-RELATED PROTEIN 8e fused to green fluorescent protein (GFP-ATG8e), we confirmed that exogenous ethanol treatment promotes autophagosome formation in vivo. Phenotypic analysis showed that deletions in the alcohol dehydrogenase gene in adh1 mutants result in attenuated submergence tolerance, decreased accumulation of ATG proteins, and diminished submergence-induced autophagosome formation. Compared to the submergence-tolerant Arabidopsis accession Columbia (Col-0), the submergence-intolerant accession Landsberg erecta (Ler) displayed hypersensitivity to ethanol treatment; we linked these phenotypes to differences in the functions of ADH1 and the autophagy machinery between these accessions. Thus, ethanol promotes autophagy-mediated submergence tolerance in Arabidopsis.

Partial Reprogramming As An Emerging Strategy for Safe Induced Cell Generation and Rejuvenation

2019 ◽  
Vol 19 (4) ◽  
pp. 248-254
Author(s):  
Marianne Lehmann ◽  
Martina Canatelli-Mallat ◽  
Priscila Chiavellini ◽  
Gloria M. Cónsole ◽  
Maria D. Gallardo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Somatic Cell ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Cell Types ◽  
Cell Reprogramming ◽  
Green Fluorescent ◽  
Original Cell

Background: Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research. Objective: Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation. Methods: In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM). Results: Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter. Conclusion: Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.

Μελέτη παραγόντων βελτιστοποίησης των τεχνικών μεταφοράς και διατήρησης του αυτόλογου λίπους (fat transer)

2016 ◽  
Author(s):  
Μαργαρίτα Μουστάκη
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Adipose Derived Stem Cells ◽  
Green Fluorescent

Εισαγωγή:Ο αυτόλογος λιπώδης ιστός έχει αποδείχθηκε ένα εξαιρετικό υλικό αύξησης μαλακών μορίων. Η απομόνωση των βλαστικών κυττάρων από τον λιπώδη ιστό (Adipose Derived Stem Cells, ADSCs) μοιραία οδήγησε την έρευνα να στραφεί στη μελέτη της συνδυασμένης μεταμόσχευσης αυτόλογου λίπους και ADSCs (cell assisted lipotransfer-CAL).Σκοπός:Η συγκεκριμένη μελέτη είναι μια in vivo μελέτη σε ζωικό πρότυπο της υποβοηθούμενης με ADSCs λιπομεταφοράς. Σκοπός της μελέτης είναι να αποδείξει με ποσοτικές μετρήσεις τη δυνατότητα των ADSCs να βελτιώνουν την ποιότητα και την μακροπρόθεσμη διατήρηση του μοσχεύματος λίπους κατά τη λιπομεταφορά, συγκρίνοντας την με την παραδοσιακή λιπομεταφορά.Μέθοδοι:Για τους σκοπούς της μελέτης, βλαστικά κύτταρα απομονώθηκαν από διαγονιδιακούς μύες C57BL / 6J-GFΡ που εκφράζουν την πράσινη φθορίζουσα πρωτεΐνη (Green Fluorescent Protein, GFP) με αποτέλεσμα τα ίδια τα βλαστικά κύτταρα να φθορίζουν, γεγονός που μας επέτρεψε την παρακόλουθηση τους απεικονιστικά. Τα GFP-θετικά βλαστικά κύτταρα αφού απομονώθηκαν από το λίπος αναμείχθηκαν με τεμαχισμένο λιπώδη ιστό που συλλέχθηκε από τις βουβωνικές χώρες των C57BL / 6JolaHsd μυών και στη συνέχεια συν-εμφυτεύτηκαν σε Hsd:Athymic Nude-Foxn1nu μύες. Σε όλους τους μύες η έγχυση του ADSC-εμπλουτισμένου μείγματος έγινε στη μία πλευρά της ράχης ενώ στην αντίθετη πλευρά της ράχης πραγματοποιήθηκε η έγχυση ελέγχου (control) με ίδια ποσότητα τεμαχισμένου λιπώδους ιστού (μη εμπλουτισμένου) από τις βουβωνικές χώρες των C57BL / 6JolaHsd μυών .Η επιβίωση των εμφυτευμένων GFP-θετικών βλαστικών κυττάρων παρακολουθήθηκε με in νίνο μοριακή απεικόνιση φθορισμού 56 ημερών. Για τη συγκριτική μελέτη του ποσοστού επιβίωσης το μοσχεύματος λίπους τα μοσχεύματα αφαιρέθηκαν χειρουργικά στις 7 και στις 56 ημέρες μετά τη μεταμόσχευση, ζυγίστηκαν και υποβλήθηκαν σε ιστολογική εξέταση και ανοσοϊστοχημικό έλεγχο για τους δείκτες CD34 και Ki67.Αποτελέσματα:Τα αποτελέσματα έδειξαν καλύτερη επιβίωση του ADSC-εμπλουτισμένου λίπους σε σύγκριση με το μη εμπλουτισμένο (63% του αρχικής μάζας έναντι 33% μεγαλύτερη σε σχέση με την παραδοσιακή λιπομεταφορά,p<0.05). Η παρουσία των φθοριζόντων ADSCs μέχρι και την 56η ημέρα παρακολούθησης επιβεβαιώθηκε με την in vivo απεικόνιση φθορισμού. Η νεο-αγγειακή πυκνότητα ήταν αυξημένη στα εμπλουτισμένα με ADSCs λιπομοσχεύματα με διαφορά στατιστικά σημαντική (p<0.05)Συμπεράσματα: Ο εμπλουτισμός των λιπο-μοσχευμάτων με ADSCs είναι μια αξιόπιστη και αποτελεσματική μέθοδος βελτιστοποίησης της τεχνικής μεταφοράς αυτόλογου λίπους. Τα ex-vivo καλλιεργημένα ADSCs συμβάλλουν στη νεο-αγγείωση και αναγέννηση του ισχαιμικού ισχαιμικού λιπομοσχεύματος .

scholarly journals Prospective isolation of mouse and human hematopoietic stem cells using Plexin domain containing 2

2021 ◽  
Author(s):  
Yosuke Tanaka ◽  
Yasushi Kubota ◽  
Ivo Lieberam ◽  
Jillian L. Barlow ◽  
Josh W. Bramley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Imaging Techniques ◽  
Xenograft Model ◽  
Hematopoietic Stem ◽  
Reporter Mice ◽  
Green Fluorescent

AbstractNumerous strategies exist to isolate hematopoietic stem cells (HSCs) using complex combinations of markers and flow cytometry. However, robust identification of HSCs using imaging techniques is substantially more challenging which has prompted the recent development of HSC reporter mice. To date, none of the molecules used in these reporters have been useful for human HSC identification. Here we report that PLXDC2 is a useful marker for both mouse and human HSCs. Using a green fluorescent protein (GFP) knock-in at the Plxdc2 locus in mice (hereafter denoted as Plxdc2-GFP), we showed that Plxdc2-GFP is highly expressed in HSCs with 1 in 2.8 Plxdc2-GFP+CD150+ cells giving long-term multi-lineage reconstitution in transplantation. Moreover, we developed a novel human PLXDC2 antibody and showed that human PLXDC2+ HSCs have stronger long-term multilineage reconstitution ability compared with PLXDC2- HSCs in a xenograft model. Thus, our study identifies PLXDC2 as a highly relevant molecule in HSC identification, potentially allowing greater purity and live in vivo tracking of these cells.SummaryTo date, few molecules are available for isolation of HSCs across species. The present study shows that PLXDC2 is a highly useful molecule for isolation of HSCs, which works across mouse and human.

Neural Development by Transplanted Human Embryonal Carcinoma Stem Cells Expressing Green Fluorescent Protein

2005 ◽  
Vol 14 (6) ◽  
pp. 339-351 ◽  
Author(s):  
R. Stewart ◽  
M. Lako ◽  
G. M. Horrocks ◽  
S. A. Przyborski
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Neural Development ◽  
Molecular Mechanisms ◽  
Embryonal Carcinoma ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Green Fluorescent

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.

scholarly journals α-Tubulin Regulates the Fate of Germline Stem Cells in Drosophila Testis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaoqian Tao ◽  
Yunqiao Dou ◽  
Guangyu Huang ◽  
Mingzhong Sun ◽  
Shan Lu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Germline Stem Cells ◽  
Intrinsic Factors ◽  
Male Germline ◽  
Evolutionarily Conserved ◽  
Drosophila Testis ◽  
Reporter Assays ◽  
Green Fluorescent

AbstractThe Drosophila testis provides an exemplary model for analyzing the extrinsic and intrinsic factors that regulate the fate of stem cell in vivo. Using this model, we show that the Drosophila αTub67C gene (full name αTubulin at 67C), which encodes α4-Tubulin (a type of α-Tubulin), plays a new role in controlling the fate of male germline stem cells (GSC). In this study, we have found that Drosophila α4-Tubulin is required intrinsically and extrinsically for GSCs maintenance. Results from green fluorescent protein (GFP)-transgene reporter assays show that the gene αTub67C is not required for Dpp/Gbb signaling silencing of bam expression, suggesting that αTub67C functions downstream of or parallel to bam, and is independent of Gbb/Dpp-bam signaling pathway. Furthermore, overexpression of αTub67C fails to obviously increase the number of GSC/Gonialblast (GB). Given that the α-tubulin genes are evolutionarily conserved from yeast to human, which triggers us to study the more roles of the gene α-tubulin in other animals in the future.

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