Timing of Wnt Inhibition Modulates Directed Differentiation of Medial Ganglionic Eminence Progenitors from Human Pluripotent Stem Cells

In vitro differentiation of human pluripotent stem cell into relevant cell types is a desirable model system that has the human biological context, is a renewable source, and is scalable. GABA interneurons and basal forebrain cholinergic neurons, derivates of the medial ganglionic eminence (MGE), are implicated in diverse neuropsychiatric diseases. Various protocols have been proposed to generate MGE progenitors: the embryoid body- (EB-) based rosette-derived (RD), the adherent (AdD), and the nonadherent (NAdD) approaches. While Wnt inhibition is frequently incorporated into the strategy, the timing varies between protocols and there is a lack of standardized outcome reporting, which precludes direct comparison. Here, we report a head-to-head comparison in three distinct experimental models to establish whether Wnt inhibition during neural stem cell, NSC (stage 1), or neural progenitor cell, NPC (stage 2), formation facilitates MGE differentiation. Wnt inhibition at both stages promotes MGE progenitor differentiation when compared to no inhibition. However, NSC (stage 1) Wnt inhibition markedly reduces the number of MGE progenitors available for downstream applications in the RD and the NAdD protocols due to early inhibition of proliferation. NPC (stage 2) Wnt inhibition in the adherent system is comparable to the EB-based methods offering a techically less challenging alternative.

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Interneuron origins in the embryonic porcine medial ganglionic eminence

10.1101/2020.12.18.423526 ◽

2020 ◽

Author(s):

Mariana L. Casalia ◽

Tina Li ◽

Harrison Ramsay ◽

Pablo J. Ross ◽

Mercedes F. Paredes ◽

...

Keyword(s):

Expression Patterns ◽

Normal Brain ◽

Female Rat ◽

Medial Ganglionic Eminence ◽

Ganglionic Eminence ◽

Embryonic Mouse ◽

Migratory Capacity ◽

Normal Brain Function ◽

Transcription Factor Expression

Interneurons contribute to the complexity of neural circuits and maintenance of normal brain function. Rodent interneurons originate in embryonic ganglionic eminences, but developmental origins in other species are less understood. Here, we show that transcription factor expression patterns in porcine embryonic subpallium are similar to rodents, delineating a distinct medial ganglionic eminence (MGE) progenitor domain. On the basis of Nkx2.1, Lhx6 and Dlx2 expression, in vitro differentiation into neurons expressing GABA and robust migratory capacity in explant assays, we propose that cortical and hippocampal interneurons originate from a porcine MGE region. Following xenotransplantation into adult male and female rat hippocampus, we further demonstrate that porcine MGE progenitors, like those from rodents, migrate and differentiate into morphologically distinct interneurons expressing GABA. Our findings reveal that basic rules for interneuron development are conserved across species, and that porcine embryonic MGE progenitors could serve as a valuable source for interneuron-based xenotransplantation therapies.Significance StatementHere we demonstrate that porcine MGE, like rodents, exhibit a distinct transcriptional and pallial interneuron-specific antibody profile, in vitro migratory capacity and are amenable to xenotransplantation. This is the first comprehensive examination of embryonic pallial interneuron origins in the pig, and because a rich neurodevelopmental literature on embryonic mouse MGE exists (with some additional characterizations in other species like monkey and human) our work allows direct neurodevelopmental comparisons with this literature.

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Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice

F1000Research ◽

10.12688/f1000research.21208.1 ◽

2019 ◽

Vol 8 ◽

pp. 1983

Author(s):

Christopher Blackwood

Keyword(s):

Stem Cell ◽

Neural Stem Cell ◽

Cell Biology ◽

Stem Cell Biology ◽

Ganglionic Eminence ◽

Lateral Ganglionic Eminence ◽

Epidermal Growth ◽

Dissection Technique

Background: The neurosphere assay is a powerful tool to study neural stem cell biology. The objective of this protocol is to create a simple and rapid approach to generate neurospheres from the dorsal lateral ganglionic eminence of late embryonic (day 17) mice. This method predicts the average number of neurospheres and provides an approximation of its expected size after 7 days in vitro. Characterization of numbers and sizes will provide investigators with quantitative data to advise on the implementation of downstream applications, including immnocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro. Furthermore, we found this method yields different sizes of neurospheres. Lastly, using an anti-GFAP antibody, we confirm that these neurospheres can be used for immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of neurospheres that will be useful for further advances in the area of stem cell biology.

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Lung Cancer Stem Cell: New Insights on Experimental Models and Preclinical Data

Journal of Oncology ◽

10.1155/2011/549181 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 19

Author(s):

Caroline Rivera ◽

Sofia Rivera ◽

Yohann Loriot ◽

Marie-Catherine Vozenin ◽

Eric Deutsch

Keyword(s):

Lung Cancer ◽

Stem Cell ◽

Cancer Stem Cell ◽

Tumor Development ◽

Experimental Models ◽

Cancer Therapies ◽

Tumor Initiating Cells ◽

Preclinical Data

Lung cancer remains the leading cause of cancer death. Understanding lung tumors physiopathology should provide opportunity to prevent tumor development or/and improve their therapeutic management. Cancer stem cell (CSC) theory refers to a subpopulation of cancer cells, also named tumor-initiating cells, that can drive cancer development. Cells presenting these characteristics have been identified and isolated from lung cancer. Exploring cell markers and signaling pathways specific to lung CSCs may lead to progress in therapy and improve the prognosis of patients with lung cancer. Continuous efforts in developingin vitroandin vivomodels may yield reliable tools to better understand CSC abilities and to test new therapeutic targets. Preclinical data on putative CSC targets are emerging by now. These preliminary studies are critical for the next generation of lung cancer therapies.

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Use of “MGE Enhancers” for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons

PLoS ONE ◽

10.1371/journal.pone.0061956 ◽

2013 ◽

Vol 8 (5) ◽

pp. e61956 ◽

Cited By ~ 20

Author(s):

Ying-Jiun J. Chen ◽

Daniel Vogt ◽

Yanling Wang ◽

Axel Visel ◽

Shanni N. Silberberg ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Embryonic Stem ◽

Medial Ganglionic Eminence ◽

Ganglionic Eminence ◽

Selection Of

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Mesenchymal stem cell derived exosomes enhance lymphangiogenesis via exosomal transfer of Ang-2/Tie2

10.1101/466987 ◽

2018 ◽

Author(s):

Ting Zhao ◽

Zhixin Yan ◽

Jinwen Liu ◽

Hui Sun ◽

Yifei Chen ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Lymphatic Vessels ◽

Tube Formation ◽

Experimental Models ◽

Organ Injury ◽

Human Umbilical Cord ◽

Beneficial Effects ◽

Membrane Bound

AbstractMesenchymal stem cell derived exosomes (MSC-Ex) are nanosized membrane-bound extracellular vesicles found in MSC conditioned medium, that have yielded beneficial effects in several experimental models of organ injury. However, the therapeutic value and mechanism of MSC-Ex in lymphedema is poorly understood. Here we find that human umbilical cord MSCs derived exosomes (hucMSC-Ex) treatment contributed to the regeneration of LYVE-1 positive lymphatic vessels and reduction of lymphedema in a mouse model of tail lymphedema. Following uptake, exosomal lymphangiogenic factors (angiopoietin (Ang)-2 and Tie2) are taken up by HDLECs and promoted HDLECs proliferation, migration, and tube formation in vitro. We also find that exosomal Ang-2 and Tie2 exert a prolymphangiogenic effect on HDLECs through upregulating Prox1 and VEGFR3/p-Akt expression. In conclusion, our result unravel a previously unappreciated prolymphangiogenic role of hucMSC-Ex in lymphedema therapy and provide a new mechanism of Ang-2 in therapeutic lymphangiogenesis.

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Sonic hedgehog contributes to oligodendrocyte specification in the mammalian forebrain

Development ◽

10.1242/dev.128.4.527 ◽

2001 ◽

Vol 128 (4) ◽

pp. 527-540 ◽

Cited By ~ 5

Author(s):

S. Nery ◽

H. Wichterle ◽

G. Fishell

Keyword(s):

Sonic Hedgehog ◽

Retroviral Vectors ◽

Medial Ganglionic Eminence ◽

Wild Type ◽

Ganglionic Eminence ◽

Early Expression ◽

Pdgfr Alpha

This study addresses the role of Sonic hedgehog (Shh) in promoting the generation of oligodendrocytes in the mouse telencephalon. We show that in the forebrain, expression of the early oligodendrocyte markers Olig2, plp/dm20 and PDGFR(alpha) corresponds to regions of Shh expression. To directly test if Shh can induce the development of oligodendrocytes within the telencephalon, we use retroviral vectors to ectopically express Shh within the mouse embryonic telencephalon. We find that infections with Shh-expressing retrovirus at embryonic day 9.5, result in ectopic Olig2 and PDGFR(alpha) expression by mid-embryogenesis. By postnatal day 21, cells expressing ectopic Shh overwhelmingly adopt an oligodendrocyte identity. To determine if the loss of telencephalic Shh correspondingly results in the loss of oligodendrocyte production, we studied Nkx2.1 mutant mice in which telencephalic expression of Shh is selectively lost. In accordance with Shh playing a role in oligodendrogenesis, within the medial ganglionic eminence of Nkx2.1 mutants, the early expression of PDGFR(alpha) is absent and the level of Olig2 expression is diminished in this region. In addition, in these same mutants, expression of both Shh and plp/dm20 is lost in the hypothalamus. Notably, in the prospective amygdala region where Shh expression persists in the Nkx2.1 mutant, the presence of plp/dm20 is unperturbed. Further supporting the idea that Shh is required for the in vivo establishment of early oligodendrocyte populations, expression of PDGFR(alpha) can be partially rescued by virally mediated expression of Shh in the Nkx2.1 mutant telencephalon. Interestingly, despite the apparent requirement for Shh for oligodendrocyte specification in vivo, all regions of either wild-type or Nkx2.1 mutant telencephalon are competent to produce oligodendrocytes in vitro. Furthermore, analysis of CNS tissue from Shh null animals definitively shows that, in vitro, Shh is not required for the generation of oligodendrocytes. We propose that oligodendrocyte specification is negatively regulated in vivo and that Shh generates oligodendrocytes by overcoming this inhibition. Furthermore, it appears that a Shh-independent pathway for generating oligodendrocytes exists.

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LdrB Toxin with In Vitro and In Vivo Antitumor Activity as a Potential Tool for Cancer Gene Therapy

Cancers ◽

10.3390/cancers11071016 ◽

2019 ◽

Vol 11 (7) ◽

pp. 1016 ◽

Cited By ~ 1

Author(s):

Yaiza Jiménez-Martínez ◽

Carmen Griñán-Lisón ◽

Hoda Khaldy ◽

Ana Martín ◽

Alba Cambrils ◽

...

Keyword(s):

Breast Cancer ◽

Gene Therapy ◽

Side Effects ◽

Lethal Effect ◽

Cancer Gene Therapy ◽

Experimental Models ◽

Cancer Gene ◽

Inhibition Of Proliferation

Due to the high prevalence of cancer in recent years, it is necessary to develop new and more effective therapies that produce fewer side effects. Development of gene therapy for cancer based on the use of suicide genes that can damage the tumor cell, without requiring a prodrug for its lethal effect, is one of the recent foci of gene therapy strategies. We evaluated the cytotoxic impact of the LdrB toxin from Escherichia coli k12 as a possible tool for cancer gene therapy. For that, colorectal and breast cancer cells were transfected under the control of a TRE3G promoter inducible by doxycycline. Our results showed that ldrB gene expression induced a drastic inhibition of proliferation in vitro, in both 2D and 3D experimental models. Moreover, unlike conventional chemotherapy, the ldrB gene induced a severe loss of proliferation in vivo without any side effects in our animal model. This antitumor outcome was modulated by cell cycle arrest in the G0/G1 phase and apoptotic death. Scanning electronic microscopy demonstrates that the LdrB toxin conserves its pore-forming ability in HCT-116 cells as in E. coli k12. Taken together, our results provide, for the first time, a proof of concept of the antitumor capacity of the ldrB gene in colorectal and breast cancer.

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Targeting miRNA-551b, a "Stemness"-like microRNA, to Eradicate AML (Stem) Cells

Blood ◽

10.1182/blood-2018-99-116024 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1494-1494

Author(s):

Tània Martiáñez ◽

Noortje Van Gils ◽

David Christian De Leeuw ◽

Eline Vermue ◽

Arjo Rutten ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Inhibition Of Proliferation ◽

Novel Target

Abstract Despite high complete remission (CR) rates achieved after chemotherapy, only 30-40% of patients with Acute Myeloid Leukemia (AML) survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy-resistant leukemia cells with stem cell properties, named "leukemic stem cells" (LSCs). LSCs use a variety of mechanisms to resist chemotherapy and targeting them is one of the major challenges in AML treatment. Since miRNAs can target multiple genes/pathways simultaneously, their modulation (downregulation or upregulation) may have great potential for the successful elimination of therapy-resistant leukemic (stem) cells (Martiañez Canales et al. Cancers 2017). Here, we show that miRNA-551b, previously identified by us as a stem cell-like miRNA, can be a potential novel target to specifically eradicate AML stem-like cells. Aiming at identification of miRNA-based therapy to specifically eradicate LSCs, while sparing normal Hematopoietic Stem Cells (HSCs), we determined expression of miRNAs in normal HSCs, Leukemic Stem Cells (LSCs) and leukemic progenitors (LP) all derived from the same AML patient's bone marrow. Using this approach, we identified miRNA-551b as being highly expressed in normal HSCs residing both in healthy and AML bone marrows. In AML, high expression of miR551b demonstrated to be associated with an adverse prognosis. Moreover, miRNA-551b was highly expressed in immature AML cases and its expression in a cohort of patients coincided with the expression of stem cell genes (De Leeuw et al. Leukemia 2016). To further elucidate the link between miRNA-551b and AML "stemness" and to test whether downregulation of miRNA-551b affects the survival of AML (stem/progenitor) cells, proliferation and the balance between differentiation and "stemness", we reduced miRNA-551b expression, either by lentiviral transduction of antagomirs or by adding locked nucleotide acid (LNA)-oligonucleotides to AML cell lines and primary AML cells. Downregulation of miRNA-551b in the stem cell-like AML cell line KG1a led to inhibition of cell growth in vitro, which was due to inhibition of proliferation rather than induction of apoptosis. KG1a tumor growth in an in vivo mouse model was also reduced when miRNA-551b was downregulated. In primary AML, miRNA-551b knockdown resulted in a significant decrease in the survival of leukemic progenitors and LSCs, while hematopoietic stem cells (HSCs) and normal progenitors from healthy bone marrows were not affected. These results suggest that a therapeutic approach inhibiting miRNA-551b expression might specifically eradicate leukemic progenitors and LSCs from primary AML, while sparing HSCs. We are currently studying miRNA-551b targets which can be responsible for this specific LSCs elimination. In conclusion, our results suggest that inhibition of miRNA-551b could be a promising approach to eliminate stem cell-like AML cells, thereby decreasing relapse rates and improving AML treatment outcome. Disclosures Ossenkoppele: Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.

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Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential

Development ◽

10.1242/dev.128.22.4597 ◽

2001 ◽

Vol 128 (22) ◽

pp. 4597-4604

Author(s):

Rita C. R. Perlingeiro ◽

Michael Kyba ◽

George Q. Daley

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Embryoid Body ◽

Embryonic Stem ◽

Cell Types ◽

Clonal Analysis ◽

Erythroid Cell ◽

Hematopoietic Stem

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (β-H1) globin, donor-derived erythroid cells in the recipient express only adult (β-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.

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The Ultrastructural Analysis of Human Colorectal Cancer Stem Cell-Derived Spheroids and Their Mouse Xenograft Shows That the Same Cells Types Have Different Ratios

Biology ◽

10.3390/biology10090929 ◽

2021 ◽

Vol 10 (9) ◽

pp. 929

Author(s):

Michela Relucenti ◽

Federica Francescangeli ◽

Maria Laura De De Angelis ◽

Vito D'Andrea ◽

Selenia Miglietta ◽

...

Keyword(s):

Colorectal Cancer ◽

Stem Cell ◽

Cancer Cells ◽

Drug Testing ◽

Flow Cytometric Analysis ◽

Experimental Models ◽

Stem Cell Marker ◽

Multicellular Spheroids

Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.

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