המחברים היו רוצים להודות ד&quot;ר Nai-Sum וונג על מתן המנגנון קאמרית היפוקסיה וגברת אליס לואי עבור התמיכה הטכנית.

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	<li>Wiliams, R. R., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation:+a+repair+strategy+for+spinal+cord+injury?.">Schwann cell transplantation: a repair strategy for spinal cord injury?.</a> Prog Brain Res. 201, 295-312 (2012).</li><li>Kanno, H., Pearse, D. D., Ozawa, H., Itoi, E., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation+for+spinal+cord+injury+repair:+its+significant+therapeutic+potential+and+prospectus.">Schwann cell transplantation for spinal cord injury repair: its significant therapeutic potential and prospectus.</a> Rev Neurosci. 26 (2), 121-128 (2015).</li><li>Lindvall, O., Kokaia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+human+neurodegenerative+disorders--time+for+clinical+translation?.">Stem cells in human neurodegenerative disorders--time for clinical translation?.</a> J Clin Invest. 120 (1), 29-40 (2010).</li><li>Terzic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+Differentiation+of+Oligodendrocyte+Progenitor+Cells+From+Mouse+Induced+Pluripotent+Stem+Cells.">Directed Differentiation of Oligodendrocyte Progenitor Cells From Mouse Induced Pluripotent Stem Cells.</a> Cell Transplant. 25 (2), 411-424 (2016).</li><li>Takashima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroepithelial+cells+supply+an+initial+transient+wave+of+MSC+differentiation.">Neuroepithelial cells supply an initial transient wave of MSC differentiation.</a> Cell. 129 (7), 1377-1388 (2007).</li><li>Felling, R. 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כל המחברים של כתב היד הזה אין כל גילוי כדי להכריז.

זה חיוני כדי לשמר את &quot;גזע&quot; של MSCs לפני העשרה של אבות עצביים באמצעות תנאים מוקדמים היפוקסי ותרבות neurosphere. מניסיוננו, MSCs ריבוי יכול להיות מזוהה באופן מהימן על ידי מורפולוגיה fibroblast שלהם מוארך כמו. לעומת זאת, MSCs אשר אימצו מורפולוגיה מרובעת יותר, מרובעת, עם סיבי הלחץ הבו...

השתלת אבות עצביים ונגזרותיהם מדגימה את ההבטחה כאסטרטגיה טיפולית בעקבות פגיעה עצבית טראומטית 1 , 2 ונוירוגנרטיון 3 , 4 . לפני היישום הקליני, זה חיוני כדי להבטיח: א) שיטה לגישה והרחבה על מקור אוטולוגי של גזע / תאים אב ו 2) אמצעי לכוון אותם סוגי תאים רלוונטיים, בוגרת 3 . ההתעניינות שלנו בטיפול בתאי פציעה בחוט השדרה הובילה אותנו לחפש מקור תא אוטולוגי חזק ואבולוצי של אבות עצביים ברקמות של מבוגרים. תת אוכלוסיות של MSC מקורן בכף עצבי, והוא נגיש בקלות מחלל המפרק. תאים אלה הם אבות עצביים שיכולים ליצור נוירונים וגליה 5 . מודלים של בעלי חיים של איסכמיה מוחית מראים כי היפוקסיה מקדמת את ההרגעה<html> Iferation ו multipotency של אבות עצביים בתוך המוח 6 . זה היה הבסיס לשימוש התנאי היפוקסי כאמצעי להרחבת על אבות עצביים הנגזרים על מוח. השתלת תאי Schwann לתוך חוט השדרה הפצוע מקדם התחדשות 2 . SCLCs ניתן לייצר MSCs באמצעות תוספת עם גורמים gliogenic ( כלומר, β-Heregulin, bFGF, ו PDGF-AA), אבל להפגין יציבות פנוטיפית. עם הנסיגה של גורמי גדילה, הם לחזור פנוטיפ דמוי fibroblast 7 . אי-יציבות פנוטיפית אינה רצויה בהשתלות תאים בשל הסיכון של הבחנה חריגה וסרטן. כמו מבשרי התא Schwann קשורות עם צרורות האקסון בתוך העצב ההיקפי עובריים 8 , הובילו cLCulture coculture עם נוירונים DRG מטוהרים עובריים 7 ,התחת = &quot;xref&quot;&gt; 9. תאים בוגרת שוואן בשלבים הם גורל מחויבים להפגין פונקציה במבחנה 7 , 9 ו in vivo 10 . הפרוטוקול שלנו להעשרת אבות עצביים מ MSC הוא פשוט ויעיל ותוצאות גידול במספר התא עבור מבחני הבאים. הגזירה של תאי גורן מחויבים באמצעות פלטפורמת coculture מאפשר לימוד של הבדל גלייה ועל הדור של תאים שוואן יציבה ופונקציונלית עבור יישומים קליניים פוטנציאליים.

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">&#945;MEM</td>
			<td style="width:140px;">Sigmaaldrich</td>
			<td style="width:101px;">M4526</td>
			<td style="width:329px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Thermofisher scientific</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Thermofisher scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Biosera</td>
			<td>FB-1280/500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27</td>
			<td>Thermofisher scientific</td>
			<td>17504-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal growth factor (EGF)</td>
			<td>Thermofisher scientific</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic fibroblast growth factor (bFGF)&#160;</td>
			<td>Peprotech</td>
			<td>100-18B/100UG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nerve growth factor (NGF)&#160;</td>
			<td>Millipore</td>
			<td>NC011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet-derived growth factor-AA (PDGF-AA)</td>
			<td>Peprotech</td>
			<td>100-13A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heregulin beta-3, EGF domain (&#946;-Her)</td>
			<td>Millipore</td>
			<td>01-201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Uridine</td>
			<td>Sigmaaldrich</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Fluro-2&#39; - deoxyuridine (FDU)</td>
			<td>Sigmaaldrich</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-D-lysine (PDL)</td>
			<td>Sigmaaldrich</td>
			<td>P7886-1G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin</td>
			<td>Thermofisher scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMAX</td>
			<td>Thermofisher scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin / streptomycin (P/S)</td>
			<td>Thermofisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Express</td>
			<td>Thermofisher Scientific</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cm plate for adherent culture</td>
			<td>TPP</td>
			<td>93100</td>
			<td>Used for selection of MSCs by tissue culture adherence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate for adherent culture</td>
			<td>TPP</td>
			<td>92006</td>
			<td>Used for expansion of MSCs following passaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraLow 6-well plate for non-adherent culture</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Used for neural progenitor enrichment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>555593</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD73</td>
			<td>BD Biosciences</td>
			<td>550256</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human/rat STRO-1</td>
			<td>R&#38;D Systems</td>
			<td>MAB1038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human nestin</td>
			<td>R&#38;D Systems</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>555480</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>554895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD73</td>
			<td>BD Biosciences</td>
			<td>551123</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat nestin</td>
			<td>BD Biosciences</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD45</td>
			<td>BD Biosciences</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-S100&#946;</td>
			<td>Dako</td>
			<td>Z031101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-p75</td>
			<td>Millipore</td>
			<td>MAB5386</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-GFAP</td>
			<td>Sigmaaldrich</td>
			<td>G3893</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Class III-beta tubulin (Tuj-1)</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Human nuclei</td>
			<td>Millipore</td>
			<td>MAB1281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td>MIC-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES buffer</td>
			<td>Sigmaaldrich</td>
			<td>H4034-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

hypoxic preconditioning of marrow-derived progenitor cells as a source for the generation of mature schwann cells

כתב יד זה מתאר אמצעי להעשרתם של אבות עצביים מאוכלוסיית תאי החלב (MSC) ולאחר מכן להפנותם אל גורל התא הבוגר של שוואן. ערכנו חולדות וחולי סרטן אנושיים לתנאים היפוקסיים חולפים (חמצן של 1% ל -16 שעות), ולאחר מכן התרחבות כנוירוספרה על מצע נמוך עם גורם גדילה של אפידרמיס (EGF) / תוספת גורמי גידולי פיברובלסטים בסיסיים (bFGF). Neurospheres היו seeded על פולי-D- ליזין / laminin מצופה רקמה תרבות פלסטיק ו מתורבת בקוקטייל gliogenic המכיל β-Heregulin, bFGF, ו טסיות הנגזרות גורם הצמיחה (PDGF) לייצר תאים דמויי תא שוואן (SCLCs). SCLCs היו מופנים למחויבות הגורל באמצעות coculture במשך 2 שבועות עם נוירונים השורש הגבי מטוהרים (DRG) נוצרים המתקבלים E14-15 הרות חולדה Sprague Dawley. תאים בשלה שוואן להוכיח התמדה ביטוי S100β / p75 ויכול ליצור קטעי myelin. תאים שנוצרו באופן זה יש פוטנציאל אPplications ב השתלת תאים אוטולוגיים בעקבות פגיעה בחוט השדרה, כמו גם דוגמנות המחלה.

סקירה של שלבי המפתח בפרוטוקול שלנו הוא באיור 1 . לסיכום, חולדה MSCs אנושיים נבחרים על ידי דבקות פלסטיק תרבות רקמות. MSC מורחב מותנים מראש עם היפוקסיה והם כפופים לתנאי נוירופר. Neurospheres הם מצופה מותר להבדיל SCLCs. SCLCs הם cocultured עם נוירונים DRG מטו?...

Watch this Scientific Journal Video about Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells at JoVE.com

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

תאים מוחיים סטרומליים (MSC) עם פוטנציאל עצבי קיימים בתוך מוח העצם. הפרוטוקול שלנו מעשיר אוכלוסייה זו של תאים באמצעות תנאים מוקדמים היפוקסי ולאחר מכן מפנה אותם להפוך לתאי Schwann בוגרת.

Hypoxic Preonditioning של תאים יליד מארו נגזר כמקור לדור של תאים שוואן בוגרת

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

hypoxic-preconditioning-marrow-derived-progenitor-cells-as-source-for

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Developmental Biology

7.2K Views.  The University of Hong Kong.  תאים מוחיים סטרומליים (MSC) עם פוטנציאל עצבי קיימים בתוך מוח העצם. הפרוטוקול שלנו מעשיר אוכלוסייה זו של תאים באמצעות תנאים מוקדמים היפוקסי ולאחר מכן מפנה אותם להפוך לתאי Schwann בוגרת. 

Video: Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

גזירה של תאי לב ותאים מתאי גזע עובריים

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

אינדוקציה ישירה של תאי גזע עצביים אדם מתאי הדם היקפי Hematopoietic אב

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

דור של תאי גזע שומן האדם דרך Dedifferentiation של מבוגרים adipocytes בתרבויות תקרה

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

מבחני מושבת Vitro לאפיון ובתאי Tri-עצמה מבודדת מלבלב Murine למבוגרים

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

אופקי הר שלם: עיבוד רומן פרוטוקול הדמיה על רקמה עבה, תלת מימדי חתכים של העור

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Photoconversion תא בודד בהמחיה שלם דג זברה

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

ניתוח חישובית של Germline Caenorhabditis elegans ללמוד הפצה של גרעינים, חלבונים ו את שלד התא

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

בדור חוץ גופית של Somite נגזרים מן האדם המושרה בתאי גזע Pluripotent

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

בדור מחוץ לתחום של לב העכבר שדה מסוים בתאי לב

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

הערכה של נזק חמצוני בעכבר הראשי פני השטח העינית/תאים גזע בתגובה אולטרה סגול (UV-C) נזק

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...