وأيد هذه الدراسة من قبل المعاهد الوطنية للبحوث الصحية منحة R01-DE021683-01 والمعاهد الوطنية للبحوث الصحية منحة R01-DE019434 إلى MTL. وأيد زمالة أبحاث معهد هوارد هيوز الطبي لMTCDCW من قبل فرانكلين مارتن كلية زمالة ACS البحوث، ومختبر Hagey للأطفال الطب التجديدي، وجائزة معهد بحوث صحة الطفل بجامعة ستانفورد كلية إجابات.

ملاحظة: تم الحصول على جميع عينات المرضى للموافقة المسبقة عن، وتم استعراض البروتوكولات التجريبية والمعتمدة من قبل جامعة ستانفورد مجلس المراجعة المؤسسية (بروتوكول # 2188 و # 9999). 1. عزل خلية والثقافة: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحصول على الأنسجة الدهنية تحت الجلد الإنسان من المرضى من النساء الأصحاء تمر lipoaspiration الاختيارية من البطن، الجناح، و / أو منطقة الفخذ تحت تخدير موضعي / عام. تأكد من أن مجلس المراجعة المؤسسية (IRB) الموافقة قد تم الحصول عليها عن بروتوكول لعزل ASCS من الأنسجة البشرية، واتباع احتياطات السلامة المؤسسية بينما كان يعمل مع مثل هذه المواد. </li><li style=";text-align:right;direction:rtl"> للحصول على صندوق التبرعات الخاص من الأنسجة الدهنية lipoaspirated، وغسل لأول مرة lipoaspirate ثلاث مرات مع أحجام متساوية من 1X العقيمة الفوسفات مخزنة المالحة (PBS). نضح بعناية وتجاهل الطبقة المائية أسفل. </li><li style=";text-align:right;direction:rtl"> إعداد العازلة كولاجيناز الهضم: 0.075٪ النوع الأول كولاجيناز في لهانكمحلول ملحي متوازن (HBSS). إعداد عازلة FACS: 2٪ FBS، 1٪ P188 و 1٪ القلم بكتيريا في برنامج تلفزيوني. تصفية كل من حلول باستخدام المتاحة تجاريا 0.22 ميكرون حجم المسام تدفق سريع مرشح polyethersulfone. </li><li style=";text-align:right;direction:rtl"> لهضم الأنسجة الدهنية غسلها، إضافة حجم مساو من كولاجيناز عازلة الهضم ووضع سفينة الهضم بشكل آمن في حمام مائي تهتز لمدة 60 دقيقة عند 37 درجة مئوية (حوالي 180 يهز / دقيقة). ملاحظة: فمن الأفضل لاستخدام أكبر حجم الوعاء الهضم مما هو مطلوب، وهذا يسمح للهضم القصوى خلال الهز (أي 250 مل من العازلة كولاجيناز الهضم، 250 مل من الأنسجة الدهنية غسلها في قارورة معقمة 1L). </li><li style=";text-align:right;direction:rtl"> تحييد النشاط الأنزيمي بإضافة حجم مساو من العازلة FACS والسماح للجلوس على RT لمدة 5 دقائق. وبعد ذلك، الطرد المركزي في 233 x ج لمدة 20 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> نضح بعناية وتجاهل طاف، مع الحرص على عدم تعكير صفو عالية الكثافة SVF بيليه. </li><li style=";text-align:right;direction:rtl"> resuspend الكرية في 5-10 مل من RT الأحمرخلية تحلل العازلة، استنادا إلى حجم بيليه. ترك حل للجلوس على RT لمدة 5 دقائق والطرد المركزي في 233 x ج لمدة 5 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> نضح طاف و resuspend بيليه بإضافة 5-10 مل من وسائط النمو التقليدي، استنادا إلى حجم بيليه (Dulbecco لتعديل النسر متوسطة [DMEM] / 10٪ FBS / 1٪ البنسلين الستربتومايسين حل [القلم بكتيريا]). </li><li style=";text-align:right;direction:rtl"> تعليق تصفية من خلال 100 ميكرون خلية النايلون مصفاة لإزالة الحطام الخلوي. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي في 233 x ج لمدة 5 دقائق عند 4 درجات مئوية، وتجاهل طاف دون الإخلال بيليه. </li><li style=";text-align:right;direction:rtl"> resuspend الكرية في 5-10 مل من وسائط النمو التقليدي، استنادا إلى حجم بيليه. </li><li style=";text-align:right;direction:rtl"> ضع 2 × 10 6 خلايا في طبق ثقافة 15 سم القياسية وإقامة الثقافات الأولية O / N عند 37 درجة مئوية / 21٪ O 2، 5٪ CO 2. الحفاظ على المستويات دون متموجة لمنع التمايز عفوية في 37 ° C / 21٪ O 2، 5٪ CO 2 في وسائل الاعلام النمو. كثافة خلية في كامل ملاحظة:متموجة 15 سم لوحة ما يقرب من 4-6 × 10 6 خلايا. </li></ol> 2. تلطيخ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ASCS الثقافة في متوسطة النمو التقليدي عند 37 درجة مئوية / 21٪ O 2، 5٪ CO 2، لمدة 36 ساعة قبل تلطيخ / الفرز. </li><li style=";text-align:right;direction:rtl"> ASCS رفع من لوحات ثقافة استخدام Accutase (وفقا للبروتوكول الشركة المصنعة). </li><li style=";text-align:right;direction:rtl"> غسل خلايا مرة واحدة مع FACS العازلة (PBS مع 2٪ FBS و 1٪ القلم بكتيريا). </li><li style=";text-align:right;direction:rtl"> الطرد المركزي في 233 x ج لمدة 5 دقائق في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> تجاهل طاف دون الإخلال بيليه. resuspend الخلايا في 0.5-1 مل من العازلة FACS (اعتمادا على عدد الخلايا وكمية من الأجسام المضادة المطلوبة). ملاحظة: حجم التعليق الخلية، وفقا لتوصيات الشركة المصنعة الأجسام المضادة FACS، ويحدد تركيز الأجسام المضادة. ومع ذلك، المعايرة تركيز الأجسام المضادة، وذلك باستخدام تركيز الانطلاق من 1: 100، وينصح عادة إذا كان هذا هو المرة الأولى لاستخدام الأجسام المضادة. </li><li style=";text-align:right;direction:rtl"> عد العدد الإجمالي للالخلايا باستخدام عدادة الكريات. </li><li style=";text-align:right;direction:rtl"> الاحتياطي 100 UL مأخوذة في 1.5 مل أنابيب الطرد المركزي التي تستخدم في عينة غير ملوثين والضوابط أحادية اللون (وفقا لعدد من الأجسام المضادة الفلورسنت المستخدمة). </li><li style=";text-align:right;direction:rtl"> إضافة الاجسام المضادة المناسبة fluorescently المسمى (بتركيزات محددة سلفا، وفقا لتعليمات الشركة المصنعة)، واحتضان لمدة 20-30 دقيقة على الجليد، محمية من الضوء. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتسمية حيوانية عظمي المنشأ: تمييع مكافحة CD90 (APC CD90 مكافحة الإنسان (Thy1)) أو مكافحة CD105 (FITC مكافحة الإنسان CD105 (endoglin)) في المخزن FACS إلى تركيز 01:50. </li><li style=";text-align:right;direction:rtl"> لتسمية السكان الخلية الأخرى (على سبيل المثال، المكونة للدم والخلايا البطانية): تمييع مكافحة CD45 (مكافحة CD45 الإنسان والمحيط الهادئ الأزرق)، ومكافحة CD34 (مكافحة الإنسان CD34 APC)، و / أو مكافحة CD31 (مكافحة CD31 الإنسان PE ) في FACS العازلة إلى تركيز 01:50. </li><li style=";text-align:right;direction:rtl"> إذا لم تكن تستخدم الأجسام المضادة مترافق مباشرة، واستخدام الأجسام المضادة الأولية المعقدة البيروكسيديز مع-streptavidin مترافق الثاني المناسبةالأجسام المضادة آرى، مثل Streptavidin PE-Cy7، في التخفيف من 1: 100. </li></ol></li><li style=";text-align:right;direction:rtl"> بعد مرور فترة الحضانة، وغسل الخلايا مرتين: ملء الأنابيب مع FACS العازلة وأجهزة الطرد المركزي في 233 x ج لمدة 5 دقائق في 4 درجات مئوية، وطاف الشفط. </li><li style=";text-align:right;direction:rtl"> resuspend الخلايا في 400-500 ميكرولتر FACS العازلة ومرشح عينات من خلال 70 ميكرون خلية النايلون مصفاة لإزالة كتل الخلية. </li><li style=";text-align:right;direction:rtl"> نقل الخلايا المسمى في أنابيب FACS مع كبار تصفية والاحتفاظ بعينات على الجليد لمدة التحليل. </li></ol> 3. تنشيط الإسفار الفرز خلية ملاحظة: الخطوات التالية ولاية معرفة سابقة في الخلية مضان تنشيط الفرز (FACS) أو مساعدة من أحد الفنيين المهرة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام برنامج FACS المناسبة والمؤامرات تحليل تصميم لدراسة مبعثر إلى الأمام (FSC)، والجانب مبعثر (SSC)، فيكوإيريترين (PE) -Texas الأحمر، الأزرق المحيط الهادئ، فلوريسئين ثيوسيانات (FITC)، Allophycocyanin (APC) وأي استخدام fluorophore أخرىد لتسمية الخلايا. </li><li style=";text-align:right;direction:rtl"> قبل تحميل في فارز الخلية، لفترة وجيزة دوامة كل عينة إلى resuspend الخلايا. </li><li style=";text-align:right;direction:rtl"> تبدأ مع تحليل العينة غير ملوثين من أجل وضع بوابات بالنسبة لمجموع السكان الخلية، لاستبعاد الحطام الخلوي، وتحديد خط الأساس تألق ذاتي. </li><li style=";text-align:right;direction:rtl"> إضافة يوديد propidium (PI) على عينة غير ملوثين وتحليلها من أجل رؤية السكان من الخلايا الحية داخل مجموع السكان الخلية. بناء بوابة حول هذه الفئة من السكان. </li><li style=";text-align:right;direction:rtl"> تحليل عينة سيطرة أحادية اللون لكل تألقي من أجل وضع تعويض مضان. </li><li style=";text-align:right;direction:rtl"> تحليل العينة الملون لتحديد موقف من البوابات للفرز. </li><li style=";text-align:right;direction:rtl"> إضافة إلى PI العينة الملون من أجل وصمة عار الخلايا الميتة. </li><li style=";text-align:right;direction:rtl"> فرز السكان الخلية المسمى. ترتيب إما إلى 1.5 مل أنابيب الطرد المركزي أو أنابيب مخروطية (15 مل) التي تحتوي على مستنبت. </li><li style=";text-align:right;direction:rtl"> وقف الفرز مرة واحدة وقد تم الحصول على الكمية المطلوبة من خلايا يدويا. </li><li style=";text-align:right;direction:rtl"> لناFACS جي، إجراء فحص النقاء من خلال تحليل جزء صغير (على سبيل المثال، 500 خلية) من سكان الخلية المكتسبة. ملاحظة: هذه الخطوة تؤكد على نقاء السكان الخلية فرزها. من الناحية المثالية، ينبغي أن يكون النقاء أكبر من 90-95٪. </li><li style=";text-align:right;direction:rtl"> خلايا الطرد المركزي في 233 x ج لمدة 5 دقائق في 4 درجات مئوية التالية مباشرة الانتهاء من الفرز وresuspend في 1 مل من المتوسط ​​الطازجة تحتوي على 10٪ FBS. ملاحظة: يمكن زيادة كمية سائل الإعلام المستخدمة على أساس حجم الخلية بيليه. </li><li style=";text-align:right;direction:rtl"> لوحة على لوحات الجيلاتين المغلفة لتحسين بقاء الخلية. اعتمادا على محصول خلية النهائي، لوحة 300،000 الخلايا لكل بئر في لوحة 6 جيدا، 100،000 الخلايا لكل بئر في لوحة 12-جيدا. </li></ol>

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T., 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=CD90+(Thy-1)+Positive+Selection+Enhances+Osteogenic+Capacity+of+Human+Adipose-Derived+Stromal+Cells.">CD90 (Thy-1) Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells.</a> Tissue Eng Part A. , (2012).</li></ol>

أي من الكتاب لديهم مصلحة مالية في أي من المنتجات والأجهزة وأو المخدرات المذكورة في هذه المخطوطة. أي من الكتاب لديه أي مصلحة مالية المتنافسة لتقديم تقرير.

حاليا، وعزل مجموعات سكانية فرعية متجانسة من ASCS من صندوق التبرعات الخاص من الأنسجة الدهنية لجسم الإنسان لا يزال يشكل تحديا على الرغم من الهدف المرغوب فيه. عزلة المجموعات السكانية الفرعية ASC الموالية للالمكونة للعظم أمر مرغوب فيه بشكل خاص، كما مثل هذه الخلايا يمكن است...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

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isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

باستخدام CD90 كعلامة للخلايا مع تعزيز النتائج تكون العظم في عزلة من سكان عالي التخصيب من ASCS الإنسان (الشكل 1A، 1B). كانت ملطخة ASCS مع باسيفيك بلو-مترافق CD45 مكافحة الإنسان، FITC مترافق CD105 مكافحة الإنسان، وAPC-مترافق CD90 مكافحة الإنسان. بعد الفرز، وكان مستوى من النقاء أ?...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

العزلة وإثراء الإنسان الدهنية المشتقة من خلايا انسجة لتعزيز تكون العظم

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

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Developmental Biology

11.4K Views.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Video: Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

العزلة وتمايز الخلايا الجذعية الدهنية المشتقة من الخنزير تحت الجلد الدهنية الأنسجة

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

الفصل Immunomagnetic من Sca1 الدهون مستودع محددة<sup&gt; عالية</sup&gt; الدهنية المشتقة من الخلايا الجذعية (مخولا لصيانة طائرات)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

العزل والتوسع في الجذعية الوسيطة / خلايا انسجة مشتقة من المشيمة البشرية الأنسجة

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

مكون للغضروف التمايز تحريض الخلايا الجذعية الدهنية المشتقة من الجاذبية الطرد المركزي

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

عزل السريع لBMPR-IB + الدهنية المشتقة الخلايا اللحمية للاستخدام في قبي عيب شفاء نموذج

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

عزل ماوس بطانة الرحم طلائي واللحمية خلايا ل<em&gt; في المختبر</em&gt; Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

عزل وتوصيف الخلايا الجذعية الوسيطة اللحمية من الحبل السري البشري والجنين المشيمة

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

العزلة وإثراء الكبد السلف مجموعات فرعية تم تحديدها من قبل رواية السطح علامة الجمع

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

العزلة، والثقافة، وتمايز الخلايا اللحمية في النخاع العظمى وفروعه اوستيوكلاست من الفئران

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

عزل وتوصيف الخلايا اللحمية الوسيطة القلب من عينات مرضى اعتلال عضلة القلب أرهيثموجينيك بيوبتيك اندوميوكارديال

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...