Establishment of functional epithelial organoids from human lacrimal glands

AbstractBackgroundTear deficiency due to lacrimal gland (LG) dysfunction is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue; however, the number of stem cells in the LG tissue is low, and 2D long-term cultivation reduces the differentiation capacity of stem cells. Nevertheless, 3D LG organoids could be an alternative for a DED therapy because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. Here, we report the development of LG organoids and their application as cell therapeutics.MethodsDigested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, specific marker expression, and histological characteristics were analyzed to authenticate the formation of LG organoids. The secretory function of LG organoids was confirmed through calcium influx or proteomics analysis after pilocarpine treatment. To explore the curability of the developed organoids, mouse-derived LG organoids were fabricated and transplanted into the lacrimal tissue of a mouse model of DED.ResultsThe histological features and specific marker expression of LG organoids were similar to those of normal LG tissue. In the pilocarpine-treated LG organoid, levels of internal Ca2+ions and β-hexosaminidase, a lysosomal protein in tear fluid, were increased. In addition, the secreted proteins from pilocarpine-treated lacrimal organoids were identified through proteomics. More than 70% of the identified proteins were proven to exosome through gene ontology analysis. These results indicate that our developed organoid was pilocarpine reactive, demonstrating the function of LG. Additionally, we developed LG organoids from patients with Sjogren’s syndrome patients (SS) and confirmed that their histological features were similar to those of SS-derived LG tissue. Finally, we confirmed that the mouse LG organoids were well engrafted in the lacrimal tissue two weeks after transplantation.ConclusionThis study demonstrates that the established LG organoids resemble the characteristics of normal LG tissue and may be used as a therapy for patients with DED.

Download Full-text

Establishment of functional epithelial organoids from human lacrimal glands

10.21203/rs.3.rs-56825/v2 ◽

2021 ◽

Author(s):

Sang Yun Jeong ◽

Woo Hee Choi ◽

Seong Gyeong Jeon ◽

Sookon Lee ◽

Jong-Moon Park ◽

...

Keyword(s):

Stem Cells ◽

Dry Eye ◽

Lacrimal Gland ◽

Culture Media ◽

Calcium Influx ◽

Eye Disease ◽

Specific Marker ◽

Gland Tissue ◽

Lysosomal Protein

Abstract Background: Tear deficiency due to dysfunction of the lacrimal gland (LG) is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue, but the number of stem cells in the LG tissue is pretty low, and 2D long-term cultivation results in the reduction of the differentiation capacity of stem cells. Whereas, 3D LG organoids could be an alternative for a DED therapeutic method, because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. In this study, we developed LG organoids and applied them as cell therapeutics. Methods: Digested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, expression of specific markers and histological characters were analyzed to prove the LG organoids. And then, the secretory function of LG organoids was confirmed by calcium influx or proteomics after pilocarpine treatment. To explore the curability of developed organoids, finally, mouse-derived lacrimal gland organoids were fabricated and transplanted into mouse lacrimal tissue with DED. Results: The histological features and specific marker expression of LG organoids were similar to normal LG tissue. In the pilocarpine treated LG organoid, internal Ca2+ ions and β -hexosaminidase as known a lysosomal protein in tear fluid was increased. Also, the secreted proteins from pilocarpine treated lacrimal organoid were identified through proteomics. More than 70% of the identified proteins were proven to exosome through GO analysis. These results indicate that our developed organoid has a reactivity to pilocarpine, which shows the function of the lacrimal gland. Additionally, we developed LG organoids from Sjogren’s syndrome patients (SS) and confirmed that their histological feutures similar to that of SS-derived LG tissue. Finally, we confirmed that organoids were well engrafted in the lacrimal tissue at 2 weeks after transplantation of mouse LG orgnoid. Conclusion: This current study demonstrate that our established lacrimal gland organoids resemble characteristics of normal lacrimal gland tissue and could be used as cell therapy for patients with dry eye syndrome.

Download Full-text

qPCR analysis of mesenchymal stem cell marker expression during the long-term culture of canine adipocyte derived stem cells

Medical Journal of Cell Biology ◽

10.2478/acb-2020-0017 ◽

2020 ◽

Vol 8 (4) ◽

pp. 139-145

Author(s):

Rut Bryl ◽

Claudia Dompe ◽

Maurycy Jankowski ◽

Katarzyna Stefańska ◽

Afsaneh Golkar Narenji ◽

...

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Stem Cell Marker ◽

Pcr Analysis ◽

Marker Genes ◽

Long Term Culture ◽

Marker Expression

AbstractDue to its availability and accessibility, adipose tissue has been the subject of various studies in many different medical fields and is believed to be a useful source of stem cells. The ability of ASCs to differentiate towards different cell lineages, with possibility of directing this differentiation, increases their possible clinical applications, and they have been widely employed in multiple therapies and treatment of different pathologies. However, a deeper understanding of the molecular mechanisms underlying the ASCs osteoblastic and chondrocyte differentiation may lead to novel applications treating a multitude of different bone-related diseases through techniques more likely meeting worldwide consensus. In this study, the RT-qPCR method was used to determine the changes in expression of ASC specific markers (CD105, CD73, CD14, CD34, CD90 and CD45) before and after long-term (14-day) in vitro cultures. To confirm the identity of the investigated cells, flow cytometry was used to evaluate the presence of positive (CD44, CD90) and negative (CD45, CD34) ASC markers. Overall, the results of the PCR analysis showed a significant change in expression of most of the marker genes, indicating significant changes in the cultured cells caused by their long-term culture, potentially altering their original stem-like characteristics.Running title: ASC marker expression during long-term in vitro culture

Download Full-text

Establishment of Functional Epithelial Organoid From Human Lacrimal Glands

10.21203/rs.3.rs-56825/v1 ◽

2020 ◽

Author(s):

Sang Yun Jeong ◽

Woo Hee Choi ◽

Sookon Lee ◽

Jong-Moon Park ◽

Mira Park ◽

...

Keyword(s):

Stem Cells ◽

Dry Eye ◽

Lacrimal Gland ◽

Culture Media ◽

Conventional Medicine ◽

Calcium Influx ◽

Gene Expression Pattern ◽

Secretory Function ◽

Organoid Culture ◽

Gland Tissue

Abstract Background: Tear deficiency due to dysfunction of the lacrimal gland is one of the major causes of dry eye disease (DED). Artificial tears as conventional medicine often alleviate symptoms, however, they insufficient to prevent the progression of severe DED. Lacrimal gland stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue, but the number of stem cells in the lacrimal gland is pretty low, and 2D long-term cultivation results in the reduction of the differentiation capacity of stem cells. Therefore, 3D lacrimal gland organoids could be an alternative for a DED therapeutic method, which is capable of prolonged growth while maintaining the characteristics of the lacrimal gland tissue.Method: Digested cells from human lacrimal gland tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, expression of specific markers and histological characters were analyzed to prove the lacrimal gland organoids. And then, the secretory function of lacrimal gland organoids was confirmed by calcium influx or proteomics after pilocarpine treatment.Result: Human lacrimal gland organoids were formed and cultured under a modified salivary organoid culture medium, and their histological features and gene expression pattern were similar to normal lacrimal gland tissue. After pilocarpine treatment, their secretory function was confirmed and the secretomes were analyzed by proteomics. More than 70% of the identified proteins were proven to exosome through GO analysis. Additionally, to explore the curability of developed organoids, mouse-derived lacrimal gland organoids were fabricated and transplanted into mouse lacrimal tissue with DED, and we confirmed that organoids were well engrafted in the lacrimal tissue.Conclusion: These results demonstrate that our established lacrimal gland organoids resemble characteristics of normal lacrimal gland tissue and could be used as cell therapy for patients with dry eye syndrome.

Download Full-text

Plxdc2 Marks Hematopoietic Stem Cells and Th2 Cytokine-Producing Innate Lymphocytes in Adult Bone Marrow

Blood ◽

10.1182/blood.v118.21.1278.1278 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1278-1278

Author(s):

Yasushi Kubota ◽

Ivo Lieberam ◽

Shinya Kimura ◽

Thomas M Jessell ◽

Shin-Ichi Nishikawa

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cell ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Cell Population ◽

Specific Marker ◽

Simple Method ◽

Hematopoietic Stem

Abstract Abstract 1278 Hematopoietic stem cells (HSCs) have been highly enriched using combinations of more than 10 surface markers. However the simple method using a few positive markers is preferable to identify HSCs location in tissue section. We performed a stringent comparative gene expression profiling analysis to find genes preferentially expressed in the HSC population, and identified a total of 63 genes that are highly expressed in HSC among various hematopoietic cell population. In order to find HSC-specific marker we focused on genes encoding cell surface protein, and found that plexin domain containing 2 (Plxdc2) is highly expressed in CD34—c-Kit+Sca-1+Lineage−(CD34−KSL) HSC population using Plxdc2::GFP knock-in mice. Only 0.2% of whole bone marrow cells were Plxdc2+, and competitive repopulation assay clearly showed that all HSCs are included in the Plxdc2+ fraction. These results identify Plxdc2 as a new marker of HSCs. Plxdc2+ population contain not only HSCs but uncharacterized c-Kitlow/−Sca-1+Lineage−cells. To further purify HSCs, we investigated the additional positive marker. Throughout the screening of various known HSC-related marker, CD150 was selected. CD150 is already recognized as a positive HSC marker (Kiel, et al. Cell 2005). The Plxdc2+CD150+ fraction represented only 0.1%±0.002% in whole bone marrow, and 6% in c-Kit+Sca-1+Lineage− cells, respectively. To test whether the combination of Plxdc2 and CD150 with or without other markers can highly enrich long-term HSCs, we competitively reconstituted irradiated mice with single Plxdc2+CD150+ cells or single Plxdc2+CD150+c-Kit+Sca-1+Lineage− cells. One out of every 4.6 Plxdc2+CD150+ cells (22%), and one out of 2.2 Plxdc2+CD150+c-Kit+Sca-1+Lineage− cells (44%) engrafted and gave long-term multi-lineage reconstitution. The simple combination of Plxdc2 and CD150 significantly increased HSC purity. In addition, we found robust levels of PLXDC2 transcripts in purified human cord blood CD34+ HSCs. Next, we attempted to characterize the another Plxdc2+ fraction which is c-Kitlow/−Sca-1+Lineage−. Multicolor flowcytometric analysis revealed that Plxdc2+c-Kitlow/−Sca-1+Lineage− cells uniformly express CD45, IL7Rα, Thy-1.2, CD27, T1/ST2 (IL1RL1, a subunit of IL33R) and CD25. These cell surface phenotype indicated that this population is probably of lymphoid lineage. However, culturing Plxdc2+ c-Kit low/−Sca-1+Lineage− cells on OP9-DL1, which supports the development of T-cell progenitors to mature T-cells, did not induce T-cell differentiation. Plxdc2+c-Kitlow/−Sca-1+Lineage−cells also did not differentiate into B cells when co-cultured with OP9 stroma cell line. Furthermore Plxdc2+c-Kitlow/−Sca-1+Lineage− cells produce IL-5 and IL-13 in response to IL-33 or a combination of IL-2 and IL-25. These characteristics resemble that of “natural helper (NH) cells”, a recently identified cell population capable of producing large amounts of Th2 cytokines in fat-associated lymphoid clusters (Moro, et al. Nature 2010). Immunohistochemical staining of bone section to detect HSCs, and functional analyses to clarify why Plxdc2 specifically express in HSCs and bone marrow “NH cells” using Plxdc2-deficient mice are our ongoing tasks. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Long-Term Single-Cell Culture of Human Pluripotent Stem Cells on Precoated rLaminin-521 Cultureware in Multiple Culture Media

Cytotherapy ◽

10.1016/j.jcyt.2016.03.250 ◽

2016 ◽

Vol 18 (6) ◽

pp. S130

Author(s):

H. Nandivada ◽

J. Montoya ◽

P. Flaherty ◽

M. Albouy ◽

B. Onteniente ◽

...

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Single Cell ◽

Pluripotent Stem Cells ◽

Culture Media ◽

Human Pluripotent Stem Cells ◽

Single Cell Culture

Download Full-text

A nomenclature for intestinal in vitro cultures

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00493.2011 ◽

2012 ◽

Vol 302 (12) ◽

pp. G1359-G1363 ◽

Cited By ~ 127

Author(s):

Matthias Stelzner ◽

Michael Helmrath ◽

James C. Y. Dunn ◽

Susan J. Henning ◽

Courtney W. Houchen ◽

...

Keyword(s):

Stem Cells ◽

Culture Media ◽

Growth Patterns ◽

In Vitro Cultures ◽

Mucosal Cells ◽

Systematic Nomenclature ◽

Number Of Publications ◽

The Moment

Many advances have been reported in the long-term culture of intestinal mucosal cells in recent years. A significant number of publications have described new culture media, cell formations, and growth patterns. Furthermore, it is now possible to study, e.g., the capabilities of isolated stem cells or the interactions between stem cells and mesenchyme. However, at the moment there is significant variation in the way these structures are described and named. A standardized nomenclature would benefit the ability to communicate and compare findings from different laboratories using the different culture systems. To address this issue, members of the NIH Intestinal Stem Cell Consortium herein propose a systematic nomenclature for in vitro cultures of the small and large intestine. We begin by describing the structures that are generated by preparative steps. We then define and describe structures produced in vitro, specifically: enterosphere, enteroid, reconstituted intestinal organoid, induced intestinal organoid, colonosphere, colonoid, and colonic organoid.

Download Full-text

The size of extracellular vesicles secreted by different types of stem cells

Nauchno-prakticheskii zhurnal «Patogenez» ◽

10.25557/gm.2018.4.9747 ◽

2018 ◽

pp. 38-42

Author(s):

И.Б. Алчинова ◽

М.В. Полякова ◽

И.Н. Сабурина ◽

М.Ю. Карганов

Keyword(s):

Stem Cells ◽

Extracellular Vesicles ◽

Culture Media ◽

Living Organism ◽

Isolation And Characterization ◽

Transmission Electron ◽

Study Results ◽

Multipotent Mesenchymal Stem Cells ◽

Rat Adipose Tissue ◽

Almost All

Механизм терапевтического действия мультипотентных мезенхимных стволовых клеток (ММСК) на облученный организм в последнее время вызывает повышенный интерес исследователей. В качестве активного участника паракринного механизма реализации этого эффекта предлагают рассматривать внеклеточные везикулы, секретируемые практически всеми клетками живого организма. Цель работы: выделить и охарактеризовать внеклеточные везикулы, продуцируемые стволовыми клетками различной природы. Материалы и методы. Суспензии внеклеточных везикул, выделенных по модифицированному протоколу дифференциального центрифугирования из культуральных жидкостей от культур ММСК костного мозга человека 2-го пассажа и ММСК жировой ткани крысы 4-го пассажа, были проанализированы методом просвечивающей электронной микроскопии и методом анализа траекторий наночастиц. Результаты. Исследование показало наличие в обоих образцах микрочастиц размерами до и около 100 нм, однако процентное содержание частиц разных размеров в суспензии различалось для двух анализируемых типов клеток. Заключение. Полученные результаты могут свидетельствовать о специфике секреции, обусловленной клеточным типом. A mechanism of the therapeutic effect of multipotent mesenchymal stem cells (MMSC) on irradiated body has recently arisen much interest of researchers. Extracellular vesicles (EVs) secreted by almost all cells of a living organism were suggested to actively contribute to the paracrine mechanism of this effect. The aim of the study was isolation and characterization of extracellular vesicles produced by various types of stem cells. Materials and methods. Suspensions of EVs were isolated from culture media of passage 2 human bone marrow-derived MMSC and passage 4 rat adipose tissue-derived MMSC using a modified protocol of differential centrifugation and then studied using transmission electron microscopy and nanoparticle tracking analysis. Results. The study showed the presence of microparticles with a size of >100 nm in the examined samples. However, the percent content of particles with different sizes in the suspension was different in two analyzed types of cell culture. Conclusion. The study results might reflect a specificity of secretion determined by the cell type.

Download Full-text