Markers for the identification of tendon-derived stem cells in vitro and tendon stem cells in situ – update and future development

AbstractGenerating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

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In situ generation of human brain organoids on a micropillar array

Lab on a Chip ◽

10.1039/c7lc00682a ◽

2017 ◽

Vol 17 (17) ◽

pp. 2941-2950 ◽

Cited By ~ 35

Author(s):

Yujuan Zhu ◽

Li Wang ◽

Hao Yu ◽

Fangchao Yin ◽

Yaqing Wang ◽

...

Keyword(s):

Stem Cells ◽

Human Brain ◽

Pluripotent Stem Cells ◽

3D Culture ◽

In Situ Generation ◽

Induced Pluripotent ◽

High Throughput Manner

We present a simple and high throughput manner to generate brain organoids in situ from human induced pluripotent stem cells on micropillar arrays and to investigate long-term brain organogenesis in 3D culture in vitro.

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The role of engineered tendon matrix in the stemness of tendon stem cells in vitro and the promotion of tendon-like tissue formation in vivo

Biomaterials ◽

10.1016/j.biomaterials.2011.05.088 ◽

2011 ◽

Vol 32 (29) ◽

pp. 6972-6981 ◽

Cited By ~ 73

Author(s):

Jianying Zhang ◽

Bin Li ◽

James H-C. Wang

Keyword(s):

Stem Cells ◽

Tissue Formation ◽

Tendon Stem Cells

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Passage-Dependent Cancerous Transformation Of Human Mesenchymal Stem Cell Of Adipose Origin During In Vitro Culture

Blood ◽

10.1182/blood.v122.21.4862.4862 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4862-4862

Author(s):

Jung Ah Kim ◽

Qute Choi ◽

Kyong Ok Im ◽

Ji Seok Kwon ◽

Si Nae Park ◽

...

Keyword(s):

Stem Cells ◽

In Vitro Culture ◽

Telomere Length ◽

Conflicts Of Interest ◽

Bac Clone ◽

Interphase Cell ◽

Cytogenetic Changes ◽

Chromosomal Changes

Abstract Introduction In vitro culture of adult human mesenchymal stem cells (hMSCs) can induce cancerous transformation, depending on environmental factors. To evaluate the passage dependent chromosomal changes of hMSCs toward malignant transformation, we passaged adipose origin hMSC to 9th passage and analyzed cytogenetic change, molecular cytogenetic changes, and telomere length variations on every passage. Methods On each passage, in situ karyotyping was performed on 3 separate batches with subsequent Giemsa staining. Karyotyping was analyzed using Metafer system (MetaSystems, Altlussheim, Germany). For analysis of nonproliferating interphase cell, each chromosome were counted with centromere fluorescent in situ hybridization (FISH) using Same Day OligoFISH™(Cellay Inc., Cambridge, Massachusetts, USA). Telomere length was analyzed using FISH technique with a Cy3-lableled Telomere peptide nucleic acid (PNA) FISH kit (DakoCytomation Denmark A/S, Glostrup, Denmark). To confirm the chromosomal translocation appeared by in situ karyotyping, we made home-brew FISH probe with bacterial artificial chromosome(BAC) clone and quantitated the proportion of abnormal cells by interphase FISH. Results On 5th passage, translocation and polysomy of chromosome 7 and 9 appeared, and on 6th passage, additional translocation t(6; 10) appeared. ISCN Karyotypes of chromosomal changes from 5th passage to 7th passage were 48,XX,+7,t(7;22)(q11.22;q13.3),+9[4]/46,XX[21] → 47,XX,+7[2]/47,XX,t(6;10)(q21;q25.1),+7[2]/46,XX[13] → 48,XX,+7,t(7;22)(q11.22;q13.3),+9[6]/ 47,XX,+7[5]/46,XX[9]. Telomere length was decreased on late passages. Fusion signal of t(7;22) on passage 5(fig 1) and that of t(6;10) on passage 6(fig 2) were confirmed by BAC clone. Conclusions The behavior of late passage (from passage 5) follows a cytogenetic profile similar to that of transformed cancer cells. Cytogenetic abnormalities which were not observed in earlier passage, showed up and disappeared, but eventually persisted during passages. We suggest in vitro environment cause hMSCs to undergo cancer-like cytogenetic changes. It is of great importance to test safeguards for clinical applications of human stem cells manufactured in vitro. Disclosures: No relevant conflicts of interest to declare.

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In vitro cell surface markers are insufficient to identify in vivo/in situ multipotent synovial mesenchymal stem cells isolated from normal or osteoarthritic knees

Osteoarthritis and Cartilage ◽

10.1016/j.joca.2017.02.057 ◽

2017 ◽

Vol 25 ◽

pp. S27-S28

Author(s):

N. Aljezani ◽

A. Affan ◽

P. Railton ◽

J. Powell ◽

R. Krawetz

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Surface ◽

Surface Markers ◽

Cell Surface Markers

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Ectopic Human Mesenchymal Stem Cell-Coated Scaffolds Create An In Vivo Leukemia Niche in NOD/SCID Mice.

Blood ◽

10.1182/blood.v114.22.559.559 ◽

2009 ◽

Vol 114 (22) ◽

pp. 559-559

Author(s):

Sarah Rivkah Vaiselbuh ◽

Morris Edelman ◽

Jeffrey Michael Lipton ◽

Johnson M. Liu

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Stem Cell Niche ◽

Scid Mice ◽

Cxcr4 Antagonist ◽

Cell Niche

Abstract Abstract 559 Introduction: Different cellular components of the normal hematopoietic niche have been identified. However, the niche for malignant hematopoiesis remains to be elucidated. Recent work of other groups has suggested that hematopoietic stem cells (HSC) within the bone marrow anchor themselves in place by attaching to osteoblasts and/or vascular sinusoid endothelial cells. We have recently identified mesenchymal stem cells (MSC) as niche-maker cells and found a crucial role of the SDF-1/CXCR4 axis in this process. Stromal Derived Factor-1 (SDF-1/CXCL12) regulates stem cell trafficking and the cell cycle via its receptor CXCR4. Methods: Polyurethane scaffolds, coated in vitro with human bone marrow MSC, were implanted subcutaneously in non-irradiated NOD/SCID mice. CD34+ HSC or primary AML cells (from a leukapheresis product) were injected either in situ or retro-orbitally in the mice and analyzed for engraftment. The mice were treated twice per week with in situ injections of SDF-1, AMD3100 (a CXCR4 antagonist) or PBS (control). After 2 to 4 weeks, the scaffolds were processed and evaluated for cell survival in the mesenchymal niche by immunohistochemistry. Results: We created in vitro MSC-coated scaffolds that retained inoculated AML cells in the presence of SDF-1, while AML cells seeded on empty scaffolds were not retained. In vivo in NOD/SCID mice, the MSC-coated scaffolds, in the presence of SDF-1 enabled homing of both in situ injected normal CD34+ HSC and retroorbital- or in situ injected primary human AML cells. The scaffolds were vascularized and showed osteoclasts and adipocytes present, suggestive of an ectopic human bone marrow microenvironment in the murine host. Finally, the SDF-1-treated scaffolds showed proliferation of the MSC stromal layer with multiple adherent AML cells, while in the AMD3100-treated scaffolds the stromal lining was thin and disrupted at several points, leaving AML cells free floating in proximity. The PBS-treated control-scaffold showed a thin single cell MSC stromal layer without disruption, with few AML cells attached. Conclusion: The preliminary data of this functional ectopic human microenvironment in NOD/SCID mice suggest that AMD3100 (a CXCR4 antagonist) can disrupt the stem cell niche by modulation of the mesenchymal stromal. Further studies are needed to define the role of mesenchymal stem cells in maintaining the hematopoietic/leukemic stem cell niche in vivo. In Vivo Leukemia Stem Cell Niche: (A) Empty polyurethane scaffold. (B)Vascularization in SQ implanted MSC-coated scaffold (s) niche in NOD/SCID mice. (C) DAB Peroxidase (brown) human CD45 positive nests of AML cells (arrows) 1 week after direct in situ AML injection. (D) Human CD45 positive myeloid cells adhere to MSC in vivo (arrows). Disclosures: No relevant conflicts of interest to declare.

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Promotion of In Vitro Chondrogenesis of Mesenchymal Stem Cells Using In Situ Hyaluronic Hydrogel Functionalized with Rod-Like Viral Nanoparticles

Biomacromolecules ◽

10.1021/acs.biomac.5b01577 ◽

2016 ◽

Vol 17 (6) ◽

pp. 1930-1938 ◽

Cited By ~ 26

Author(s):

Panita Maturavongsadit ◽

Jittima Amie Luckanagul ◽

Kamolrat Metavarayuth ◽

Xia Zhao ◽

Limin Chen ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Viral Nanoparticles

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Biology of Stem Cells in Human Umbilical Cord Stroma: In Situ and In Vitro Surveys

Stem Cells ◽

10.1634/stemcells.2006-0286 ◽

2007 ◽

Vol 25 (2) ◽

pp. 319-331 ◽

Cited By ~ 335

Author(s):

Sercin Karahuseyinoglu ◽

Ozgur Cinar ◽

Emine Kilic ◽

Fadil Kara ◽

Guvem Gumus Akay ◽

...

Keyword(s):

Stem Cells ◽

Umbilical Cord ◽

Human Umbilical Cord

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Immortalized mesenchymal stem cells: a safe cell source for cellular or cell membrane-based treatment of glioma

10.21203/rs.3.rs-382529/v1 ◽

2021 ◽

Author(s):

Yuxuan Zhang ◽

Jie Liu ◽

Yunzhao Mo ◽

Zetao Chen ◽

Taoliang Chen ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Membranes ◽

Glioma Cells ◽

Soft Agar ◽

Proliferative Capacity ◽

Soft Agar Cloning ◽

The Brain

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have emerged as putative therapeutic tools due to their intrinsic tumour tropism, anti-tumour, and immunoregulatory properties. The limited passage and self-differentiation abilities of MSCs in vitro hinder preclinical studies of mesenchymal stem cells, and makes the MSC-based treatment of tumours lack a stable, uniform, and homogeneous source of cells. In this study, we focused on the safety of immortalised mesenchymal stem cells (im-MSCs) and, for the first time, studied the feasibility of im-MSCs as candidates for the treatment of glioma.METHODS: The im-MSCs were constructed by the lentiviral transfection of genes, and the proliferative capacity of immortalised MSCs and the proliferative phenotype of MSCs and MSCs co-cultured with glioma cells (U87) were measured using CCK-8 and EdU assays. After long-term culture, karyotype analysis of im-MSCs was conducted. In addition, the tumourigenicity of engineered MSCs was evaluated using soft agar cloning assays. Moreover, the engineered cells were injected into the brain of female BALB/c nude mice. Finally, the cell membranes of im-MSCs were labelled with DIO or DIR to detect their ability to be taken by glioma cells and target in situ gliomas using an IVIS system.RESULTS: Im-MSCs expressed CD73, CD90, CD105, CD29, and CD44 but did not express CD45, CD34, CD14, CD11b, or CD31. Engineered MSCs maintained the ability to differentiate into mesenchymal lineages in vitro. Im-MSCs showed stronger proliferative capacity than unengineered MSCs without colony formation in soft agar, no tumourigenicity in the brain, and normal chromosomes. MSCs or im-MSCs co-cultured with U87 cells showed enhanced proliferation ability, but did not show malignant characteristics in soft agar cloning experiments. Immortalised cells continued to express homing molecules. The cell membranes of im-MSCs were taken up by glioma cells and targeted in situ gliomas in vivo.CONCLUSIONS: Im-MSCs are promising candidates for cellular anti-glioma therapy. Im-MSCs provide a safe, adequate, quality-controlled, and continuous source of cells or cell membranes for the treatment of glioma.

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