الكتاب أود أن تقر وأشكر هؤلاء الأفراد البحوث الإضافية التي ساهمت في تطوير بروتوكول صفها وعزلتها من ASCS، بما في ذلك: الدكتور H. بيتر لورنز، دكتوراه في الطب، الدكتور هيروشي Muzuno، دكتوراه في الطب، الدكتور جيري هوانغ، MD الدكتور آدم كاتز، دكتوراه في الطب، والدكتور وليام Futrell، دكتوراه في الطب، الدكتور رونغ تشانغ، DDS، دكتوراه، والدكتور لاريسا رودريجيز، دكتوراه في الطب، الدكتور زيني ألفونسو، دكتوراه، والدكتور جون فريزر، دكتوراه. وقد مولت النتائج المقدمة، في جزء منه، من خلال المنح البحثية المقدمة من المعاهد الوطنية للصحة، بما في ذلك NIAMS ومعاهد NIDCR.

<ol>
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واضعو هذه المخطوطة هي المخترعين المشارك على براءة اختراع تمتلكها الحكام من جامعة كاليفورنيا ومرخصة لCytori التداوي.

الأنسجة الدهنية لعزل ASCS يمكن أن يأتي في أشكال كثيرة: من قطعة صلبة من الأنسجة التي تم الحصول عليها من خلال استئصال أو رأب الشحم إلى قطع أصغر الحصول عليها عن طريق إما استخراج حقنة أو بمساعدة شفط رأب الشحم (أي شفط الدهون). ما إذا كان المزيد من الخلايا صندوق التبرعات ا?...

في عام 2001، وقد وصفت مجموعة من السكان المفترضة من الخلايا الجذعية متعددة القدرات من الأنسجة الدهنية في مجلة هندسة الأنسجة 1. أعطيت هذه الخلايا اسم معالجتها Lipoaspirate أو جيش التحرير الشعبى الصينى الخلايا بسبب اشتقاقها من الأنسجة المصنعة lipoaspirate الحصول عليها من خلال الجراحة التجميلية. واستند أسلوب العزل الموضحة في هذه المقالة على الاستراتيجيات القائمة الأنزيمية للعزلة من انسجة الوعائية الكسر (صندوق التبرعات الخاص) من الأنسجة الدهنية 2. تم تعريف صندوق التبرعات الخاص باعتباره الحد الأدنى من السكان معالجة خلايا الدم الحمراء، والخلايا الليفية، الخلايا البطانية وخلايا العضلات الملساء، pericytes وقبل الخلايا الشحمية التي لم تنضم إلى ثقافة الركيزة الأنسجة 2، 3. ويقترح زراعة هذا SVF مع مرور الوقت للقضاء على العديد من هؤلاء السكان الخلية تلويث ويؤدي إلى ملتصقة والسكان ليفية. وقد تم تحديد هذه الخلايا الليفية في الأدب على مدى السنوات ال 40 الماضية بأنها مرحلة ما قبلالخلايا الشحمية. ومع ذلك، أظهر فريق بحثنا أن هذه الخلايا تمتلك أرومية متوسطة multipotency وإعادة تسمية السكان SVF ملتصقة باسم خلايا جيش التحرير الشعبى الصينى. وأضافت دراسات لاحقة من قبل العديد من المجموعات البحثية الأخرى لهذه الإمكانات، مما يشير إلى كل من الأديم الباطن وإمكانات الأدمة (للمراجعة انظر 4). منذ ذلك الوقت، ظهرت العديد من الشروط الإضافية لهذه الخلايا في الأدب. من أجل توفير نوع من التوافق في الآراء، اعتمد مصطلح الخلايا الجذعية الدهنية المشتقة أو ASCS في المؤتمر الثاني IFATS السنوية 2. على هذا النحو، سيتم استخدام ASC المدى في هذه المقالة. بروتوكول الموصوفة في هذه المقالة هو إجراء بسيط نسبيا والتي تتطلب معدات المختبرات القياسية ويستخدم الكواشف بسيطة مثل الفوسفات مخزنة المالحة والكواشف وسائل الاعلام زراعة الأنسجة القياسية وكولاجيناز. ويمكن ان تنتج أعداد كبيرة من ASCS اعتمادا على كمية من النسيج الدهني بدءا حجم وج اللاحقةالوقت ulture. ومع ذلك، فإن معالجة مثل هذا المبلغ الكبير من الأنسجة الدهنية يمكن أن تقدم بعض القضايا المادية التي يمكن تخفيفها إلى درجة باستخدام هذا البروتوكول. وعلاوة على ذلك، وهذا البروتوكول لا تتطلب مرافق زراعة الأنسجة عقيمة واغطية وافق السلامة الأحيائية، مما يجعل من الضروري استخدام مرفق زراعة الأنسجة المعتمدة. يمكن هذا الشرط أيضا خفض فائدة السكان ASC في التطبيقات السريرية ما لم يتم عزل كانوا في الممارسات التصنيعية الجيدة مرافق (GMP) وافق المصممة لعزل وتوسيع المواد للاستخدام السريري. كبديل، فإن النظم الآلية التي يمكن عزل ASCS في نظام مغلق في غرفة العمليات تجنب هذه المسألة الأساسية والسماح للاستخدام الفوري من ASCS دون أي حاجة لاحقة التوسع في المختبر. حتى الآن، هناك ستة النظم الآلية المتوفرة تجاريا لعزل الخلايا من الأنسجة البشرية. هذه الأنظمة قد تجعل من الممكن عزلعدد كبير من ASCS من كميات كبيرة من الأنسجة الدهنية التالية الحصاد على الفور. ويمكن عندئذ أن يعيد هذه ASCS في جسم المريض لمجموعة متنوعة من الأغراض دون التجدد المريض الاضطرار إلى مغادرة غرفة العمليات. بالإضافة إلى هذا البروتوكول يصف العزلة دليل ASCS، وتعطى بروتوكول لعزل الآلي ASCS باستخدام نظام Celution أيضا في مقال مصاحب.

<table border="1">
	<tbody>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				Reagent</td>
		</tr>
		<tr>
			<td>
				DMEM (Dulbecco&#39;s Modification of Eagle&#39;s Medium)</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				10-013-CV</td>
			<td>
				with 4.5 g/ml glucose, L-glutamine, sodium pyruvate</td>
		</tr>
		<tr>
			<td>
				Penicillin/Streptomycin</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				30-002-CI</td>
			<td>
				10,000 IU/ml penicillin/10,000 &mu;g/ml streptomycin</td>
		</tr>
		<tr>
			<td>
				Amphotericin B</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				30-003-CF</td>
			<td>
				250 &mu;g/ml amphotericin B</td>
		</tr>
		<tr>
			<td>
				10X PBS (Phospho-buffered Saline)</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				25-053-CI</td>
			<td>
				without calcium, without magnesium</td>
		</tr>
		<tr>
			<td>
				Trypsin/EDTA</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				20-031-CV</td>
			<td>
				0.25 % trypsin/2.21mM EDTA</td>
		</tr>
		<tr>
			<td>
				Collagenase type IA (from Clostridium histolyticum)</td>
			<td>
				Sigma</td>
			<td>
				C2674</td>
			<td>
				crude preparation; &lt;125 collagen digestion units/mg solid</td>
		</tr>
		<tr>
			<td>
				FBS (Fetal Bovine Serum) heat inactivated</td>
			<td>
				Gemini Bioproducts</td>
			<td>
				100106</td>
			<td>
				USDA source, heat inactivated</td>
		</tr>
		<tr>
			<td>
				10 ml serological pipettes</td>
			<td>
				Genesee Scientific</td>
			<td>
				12-104</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				25 ml serological pipettes</td>
			<td>
				Genesee Scientific</td>
			<td>
				12-106</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				50 ml polypropylene centrifuge tubes</td>
			<td>
				Genesee Scientific</td>
			<td>
				21-106</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				100 mm tissue culture dishes</td>
			<td>
				Genesee Scientific</td>
			<td>
				25-202</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				150 mm tissue culture dishes</td>
			<td>
				Genesee Scientific</td>
			<td>
				25-203</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				500 ml Stericup Filter Units</td>
			<td>
				Millipore</td>
			<td>
				SCGPU05RE</td>
			<td>
				PES membrane, 0.22 &mu;m pore</td>
		</tr>
		<tr>
			<td>
				Cell strainers</td>
			<td>
				FisherBrand</td>
			<td>
				22-363-549</td>
			<td>
				100 &mu;m nylon mesh</td>
		</tr>
		<tr>
			<td>
				dexamethasone - water soluble</td>
			<td>
				Sigma</td>
			<td>
				D-2915</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				L-ascorbic-acid 2 phosphate</td>
			<td>
				Sigma</td>
			<td>
				A-8960</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				&beta;-glycerophosphate disodium salt</td>
			<td>
				Sigma</td>
			<td>
				G-9422</td>
			<td>
				also known as glycerophosphate</td>
		</tr>
		<tr>
			<td>
				insulin</td>
			<td>
				Sigma</td>
			<td>
				I-6634</td>
			<td>
				made from bovine pancreas</td>
		</tr>
		<tr>
			<td>
				indomethacin</td>
			<td>
				Sigma</td>
			<td>
				I-7378</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				apo-transferrin</td>
			<td>
				Sigma</td>
			<td>
				T-4382</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				TGF&beta;1</td>
			<td>
				R&amp;D Systems</td>
			<td>
				240-B-002</td>
			<td>
				recombinant human</td>
		</tr>
		<tr>
			<td>
				Oil Red O</td>
			<td>
				Sigma</td>
			<td>
				O-0625</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Alcian Blue</td>
			<td>
				Sigma</td>
			<td>
				A-5268</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Silver nitrate</td>
			<td>
				Sigma</td>
			<td>
				S-0319</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Hydrochloric acid</td>
			<td>
				Fisher Scientific</td>
			<td>
				A144</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Paraformaldehyde</td>
			<td>
				Fisher Scientific</td>
			<td>
				30525-89-4</td>
			<td>
				supplied as a 16 % stock</td>
		</tr>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				[header]</td>
		</tr>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				Equipment Needed</td>
		</tr>
		<tr>
			<td>
				Class II A/B Biosafety hood</td>
			<td>
				Thermo Scientific</td>
			<td>
				&nbsp;</td>
			<td>
				ensure hood has vacuum lines for aspiration</td>
		</tr>
		<tr>
			<td>
				Benchtop centrifuge</td>
			<td>
				Hermle Labnet</td>
			<td>
				Z383</td>
			<td>
				Swing-out rotor for 50 ml tubes required, capable of 1200 x g</td>
		</tr>
		<tr>
			<td>
				Water bath</td>
			<td>
				Fisher Scientific Isotemp</td>
			<td>
				S52602Q</td>
			<td>
				5-10L capacity, capable of 37 C</td>
		</tr>
		<tr>
			<td>
				Automated Pipette Aids</td>
			<td>
				Drummond Pipette Aid XL</td>
			<td>
				4-000-105</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				CO2 Incubator</td>
			<td>
				Thermo Scientific</td>
			<td>
				Forma 310</td>
			<td>
				direct heat or water jacketed</td>
		</tr>
	</tbody>
</table>

	&nbsp;

manual isolation of adipose-derived stem cells from human lipoaspirates

في عام 2001، وصف الباحثون في جامعة كاليفورنيا، لوس أنجلوس، وعزل السكان جديدة من الخلايا الجذعية البالغة من liposuctioned الأنسجة الدهنية التي أسموه في البداية خلايا Lipoaspirate المصنعة أو خلايا جيش التحرير الشعبى الصينى. منذ ذلك الحين، تم إعادة تسمية هذه الخلايا الجذعية الخلايا الجذعية الدهنية كما مشتقة أو ASCS وذهبت لتصبح واحدة من خلايا جذعية للبالغين الأكثر شعبية للسكان في مجالات أبحاث الخلايا الجذعية والطب التجديدي. وصف الآلاف من المقالات الآن استخدام ASCS في مجموعة متنوعة من النماذج الحيوانية التجدد، بما في ذلك تجديد العظام، وإصلاح الأعصاب الطرفية والهندسة القلب والأوعية الدموية. وقد بدأت المقالات الأخيرة لوصف عدد لا يحصى من الاستخدامات لASCS في العيادة. ويحدد البروتوكول هو موضح في هذه المقالة الإجراء الأساسي لليدويا وإنزيمي عزل ASCS من كميات كبيرة من lipoaspirates تم الحصول عليها من الإجراءات التجميلية. يمكن بسهولة أن تحجيم هذا البروتوكول أعلى أو لأسفل لaccommodأكلت حجم lipoaspirate ويمكن تكييفها لعزل ASCS من الأنسجة الدهنية التي تم الحصول عليها من خلال abdominoplasties وإجراءات أخرى مماثلة.

مخطط بروتوكول أعلاه يصف، طريقة الأنزيمية دليل للعزلة من صندوق التبرعات الخاص من عينة كبيرة الحجم lipoaspirate. في هذا SVF العديد من سكان الخلية، بما في ذلك ASC. العديد من الدراسات تقترح أن زراعة هذا صندوق التبرعات الخاص في ظل ظروف زراعة الأنسجة القياسية سنختار السكان الليفية ...

Watch this Scientific Journal Video about Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates at JoVE.com

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

في عام 2001، وصف الباحثون في جامعة كاليفورنيا عزل السكان من الخلايا الجذعية البالغة، ووصف الخلايا الجذعية المشتقة الدهنية أو ASCS، من الأنسجة الدهنية. توضح هذه المقالة عزلة ASCS من lipoaspirates باستخدام دليل، بروتوكول الهضم الأنزيمية باستخدام كولاجيناز.

عزل الخلايا الجذعية من دليل الدهنية المشتقة من Lipoaspirates الإنسان

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

manual-isolation-adipose-derived-stem-cells-from-human

Research

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40.9K Views.  Cytori Therapeutics Inc.  في عام 2001، وصف الباحثون في جامعة كاليفورنيا عزل السكان من الخلايا الجذعية البالغة، ووصف الخلايا الجذعية المشتقة الدهنية أو ASCS، من الأنسجة الدهنية. توضح هذه المقالة عزلة ASCS من lipoaspirates باستخدام دليل، بروتوكول الهضم الأنزيمية باستخدام كولاج...

Video: Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

مناعي لونين من الخلايا الجذعية الجنينية البشرية

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

عزل الخلايا الجذعية من الإنسان Xenografts سرطان البنكرياس

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

العزلة وثقافة الكبار الخلايا الجذعية الظهارية من الجلد البشري

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

السذاجة الكبار الخلايا الجذعية عزل من المرحلة الابتدائية الثقافات الخلايا الليفية الإنسان

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

العزلة وثقافة الأسنان طلائي الخلايا الجذعية من الكبار ماوس القاطعة

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

عزل وتكاثر وتمايز الخلايا الجذعية المشتقة من المكاك الريسوس الدهنية

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

عزل وتوصيف وتطبيق علاجي للحويصلات خارج الخلية من الخلايا الجذعية الوسيطة البشرية المستزرعة

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

عزل الخلايا الجذعية المشتقة من الأنسجة الدهنية وتوصيفها

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

تسليط الضوء على المؤلف: عزل وتحديد الخلايا الجذعية الوسيطة المشتقة من الأنسجة الدهنية لفئران Sprague Dawley  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

عزل وتحديد الخلايا الجذعية الوسيطة المشتقة من الأنسجة الدهنية لفئران Sprague Dawley

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...