この作品は、関節炎の国立研究所からの助成金と筋骨格皮膚疾患、NIH RO1AR054458によってサポートされていました。

1。 EndoremとhMSCのラベリング<ol><li>細胞は、少なくとも18時間標識の前に80％コンフルエントに増殖させる。 </li><li>この時間の間に、ラベルのメディアは、100μgの鉄/ mlの無血清培地の用量でサンプルにEndoremを加えることによって調製される。 </li><li>細胞がコンフルエントに増殖された後、培地を吸引し、細胞をPBSまたは無血清培地で1 ×洗浄する。 </li><li>次に、リンス液を吸引され、以前に作成したラベルのメディアが追加されます。次に、細胞を37℃でインキュベートする° C、4時間、5％CO 2を 。 </li><li> 4時間インキュベーションした後、メディアをラベル付けすることによって、吸引し、細胞をPBSですすぎ、いつものようにトリプシン処理されています。 </li><li>一度トリプシン、細胞をPBSで3回洗浄し、5分間400rcfで遠心分離されています。 </li><li>この洗浄ステップの後、細胞は、懸濁し数え、生存率を評価し、実験のために必要に応じて使用されています。 </li></ol> 2。アガロースの準備<ol><li>細胞が標識されている後には、アガロース溶液を準備する時間です。 </li><li>ファインスケールでは、アガロースの粉末80mgを秤量しているとPBSの2ミリリットルに追加。 </li><li>この溶液を穏やかに振とうし、オートクレーブ処理しています。 </li><li>約40分後に、流体アガロース溶液をオートクレーブから取り出すことができる。音量を測定し、予熱されたPBSで適宜調整する必要があります。 </li><li>次いで溶液を42に冷却される° C </li></ol> 3。 MASIの作成<ol><li>アガロース溶液が所望の温度に達すると、細胞は1.5ミリリットルエッペンドルフチュー​​ブ内の最後の時間をカウントし、遠心分離されています。 </li><li>遠心後、上清を捨て、細胞を30 × 10 6細胞/ mlの濃度に達するようにDMEMと混合されています。 </li><li>今細胞は42℃の温かいアガロースと混合されています。 </li><li>コー​​ルドピペットチップは、アガロースが先端の内部に固化する可能性があります、したがって、それは上下ホット滅菌PBSをピペッティングすることにより予熱するためのヒントをお勧めします</li><li>今、均一な懸濁液が作成されるようにメディアが取り上げて細胞メディア懸濁液と十分に混合されているアガロースの同じボリューム。 </li><li>アガロースが固化するのに十分クールダウンしないように混合しながら予熱した水浴内部エッペンドルフチュー​​ブを保管してください。 </li><li>懸濁液が均質になると、細胞が欠損に移植されています。 </li></ol><img src="/files/ftp_upload/1793/1793fig1.jpg" alt="図1" /> 図1。軟骨欠損に移植hMSCsと膝蓋骨試料の冠状T2強調SE像（TR 4000 msに/ TE 18.27ミリ秒）。

<ol>
	<li>Mauck, R. L., Yuan, X., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+and+functional+maturation+of+bovine+mesenchymal+stem+cells+in+long-term+agarose+culture.">Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture.</a> Osteoarthritis Cartilage. 14, 179-189 (2006).</li><li>Pittenger, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284, 143-147 (1999).</li><li>Rahfoth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+allograft+chondrocytes+embedded+in+agarose+gel+into+cartilage+defects+of+rabbits.">Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits.</a> Osteoarthritis Cartilage. 6, 50-65 (1998).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+iron+oxide+nanoparticles+for+non-invasive+in+vivo+tracking+with+MR+imaging.">Labeling hESCs and hMSCs with iron oxide nanoparticles for non-invasive in vivo tracking with MR imaging.</a> J Vis Exp. , (2008).</li></ol>

記述されているプロトコルは、酸化鉄ナノ粒子でのMSCラベルを付けたり、軟骨の欠損にこれらの標識MSCを移植する再現可能な方法を提供します。この手法は、MASIの障害の早期発見を可能にするMRイメージング、と軟骨欠損における幹細胞移植の非侵襲的な描写を可能にします。標識された細胞の転位または流出がMR画像上に移植のサイトからラベルの消失に基づいて診断することができます?...

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		<th>Comment</th>
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<td>D-MEM High Glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D5648</td>
<td>&nbsp;</td>
<tr>
<td>PBS (Ca++, Mg++ free)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA 0.05%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25399-120</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
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<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>Ferumoxides (Endorem)</td>
<td>
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<td>Guerbet</td>
<td>&nbsp;</td>
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<td>Agarose Typ VII</td>
<td>&nbsp;</td>
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<td>A9045</td>
<td>Dilute Agarose in PBS to final concentration of 4%</td>
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implantation of ferumoxides labeled human mesenchymal stem cells in cartilage defects

ティッシュエンジニアリングの分野では、特定の細胞や機能的な組織の再生に向けたエンジニアリング、細胞生物学と医学の原理を統合しています。マトリックス関連する幹細胞移植（MASI）は関節炎や外傷性関節損傷に起因する軟骨欠損を再生成することを目指しています。成体間葉系幹細胞（MSC）は、軟骨系統の細胞に分化する能力を持っていると、セルベースの​​関節軟骨の修復技術のための有望な結果が示されている。自家のMSCは、組織の様々な単離することができるが、その分化能を失うことなく、細胞培養に拡大することができる、とin vitroおよび in vivo 1、2 の軟骨細胞分化を示している。 軟骨欠損にローカル保存し、移植したMSCsの実行可能性を提供するために、足場はまた、その後の分化と増殖をサポートする、必​​要とされる。足場ガイド組織形成のアーキテクチャとは、幹細胞が産生する細胞外マトリックスは、、拡大することが可能。以前の調査では、2％アガロース足場が安定した硝子軟骨の開発をサポートする可能性がありますし、免疫応答3を誘発しないことが示されている。 MASIの移植された幹細胞の長期保存には、軟骨再生のための非常に重要です。酸化鉄ナノ粒子とMSCのラベルは、非侵襲的なMR撮像技術4をin vivoでの追跡では 、長期が可能になります。 このプレゼンテーションでは、酸化鉄ナノ粒子で標識MSCは、軟骨欠損に細胞 - アガロース構造とこれらの構造の注入を生成するためのテクニックを紹介します。標識された構造は、MRイメージングとの非侵襲的に追跡することができる。

Watch this Scientific Journal Video about Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects at JoVE.com

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

プレゼンテーションの目的は、MRイメージングで可視化することができます軟骨欠損におけるマトリックス関連する幹細胞移植を、生成するために高度に再現可能な方法を示すことです。幹細胞は、アガロースと混合のFDA承認されたFerumoxides、、軟骨欠損に移植し、7T MRスキャナで撮像で標識されています。

軟骨欠損のヒト間葉系幹細胞を標識Ferumoxidesの注入

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

implantation-ferumoxides-labeled-human-mesenchymal-stem-cells

Research

JoVE Journal

Biology

10.5K ビュー.  Medical Center, University of California San Francisco. プレゼンテーションの目的は、MRイメージングで可視化することができます軟骨欠損におけるマトリックス関連する幹細胞移植を、生成するために高度に再現可能な方法を示すことです。幹細胞は、アガロースと混合のFDA承認されたFerumoxides、、軟骨欠損に移植し、7T MRスキャナで撮像で標識されています。

ビデオ: Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

生物学

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

ファージディスプレイライブラリーからのペプチドは、間葉系細胞における遺伝子発現を調整するとUnicortical骨欠損に骨形成を増強する

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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.

成体マウス切歯から歯科上皮幹細胞の単離および培養

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

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

ヒト間葉系幹細胞から小さな細胞外小胞を単離するための簡単なベンチトップろ過法

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

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.

培養ヒト間葉系幹細胞からの細胞外小胞の単離、特性評価、および治療応用

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

脂肪組織由来幹細胞の単離と特性評価

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.

著者スポットライト:Sprague Dawleyラットの脂肪組織に由来する間葉系幹細胞の単離と同定  

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

スプレイグドーリーラットの脂肪組織由来間葉系幹細胞の単離と同定

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