成体表皮干细胞的分离培养与鉴定

目的 探讨成体表皮干细胞体外分离、培养和鉴定的技术方法。为组织工程皮肤的构建提供种子细胞。方法 通过中性蛋白酶消化成人包皮获得表皮细胞，再通过胰蛋白酶与乙二胺四乙酸（EDTA）消化并制成单个表皮细胞悬液，接种至人胎盘Ⅳ型胶原包被的培养瓶内．以Dulbecco改良Eagle培养基／F12（DMEM／F12，1：1）作为表皮干细胞培养基置培养箱内培养，37℃静置10～l5min，留用快速贴壁细胞继续培养，所得细胞分别以能否呈克隆状生长、流式细胞仪测细胞周期、透射电镜观察其超微结构及β1-整合素、角蛋白19（K19）免疫细胞化学染色进行鉴定。结果 人胎盘Ⅳ型胶原包被的培养瓶内快速贴壁细胞继续培养24h后细胞呈克隆状生长；电镜观察其超微结构示非成熟细胞特征；细胞周期分析示，第2代中静息期／DNA合成前期（G0／G1期）细胞占86．83％。β1-整合素、K19免疫细胞化学染色呈阳性。结论 成体表皮干细胞在体外得到成功分离与培养。     

Induction of hair follicle neogenesis with cultured mouse dermal papilla cells in de novo regenerated skin tissues

De novo skin regeneration with human keratinocytes amplified in culture is a life‐saving procedure for patients with extensive skin loss and chronic wounds. It also provides a valuable platform for gene function and therapeutic assessments. Nevertheless, tissues generated in this manner lack hair follicles that are important for skin homeostasis, barrier function, and repair. In this study, we generated skin tissues with human keratinocytes combined with dermal papilla (DP) cells isolated from mouse whisker hair. For this, cultured keratinocytes and mouse DP (mDP) cells were mixed at 10:1 ratio and seeded onto devitalized human dermal matrix derived from surgically discarded human abdominoplasty skin. After 1 week in submerged culture, the cell/matrix composites were grafted onto the skin wound beds of immunocompromised NSG.SCID mice. Histological analysis of 6‐week‐old skin grafts showed that tissues generated with the addition of mDP cells contained Sox2‐positive dermal condensates and well‐differentiated folliculoid structures that express human keratinocyte markers. These results indicate that cultured mDP cells can induce hair follicle neogenesis in the de novo regenerated skin tissues. Our method offers a new experimental system for mechanistic studies of hair follicle morphogenesis and tissue regeneration and provides insights to solving an important clinical challenge in generation of fully functional skin with a limited source of donor cells.     

Use of homeobox gene expression patterns to determine anatomical regions of origin for body surface tissues derived from adult mice

Anatomical regions of the skin have distinct functions and anatomical characteristics, including thicker or thinner epidermis, more or fewer hair follicles, and lighter or darker skin. For a better therapeutic outcome of skin transplantation, site‐specific characteristics of grafted tissues need to be taken into account in terms of their functionality and beauty. However, there is no method for evaluating positional information of epidermal cells. Homeobox genes are expressed along the anterior–posterior axis and direct the body plan in the animal development process. Although the expression of several HOX genes is known to be retained as the positional information in adult tissue, their expression patterns in the body surface tissues in adult mammals are still incompletely understood. In this study, we investigated the expression patterns of 40 homeobox genes, including 39 Hox genes and the paired box 6 (Pax6) gene, in body surface tissues of adult mice. On the basis of the results obtained, we proposed, for the first time, a method for determining anatomical regions of origin for body surface tissues derived from adult mice using Hox genes and Pax6. Evaluation of expression levels of at least 7 Hox genes and Pax6 should be sufficient to distinguish 11 anatomical body surface tissues derived from the adult mouse body. The proposed method may be useful not only for determining the origin of surface tissues from specific anatomical regions of the mammalian body but also for predicting positional information of epithelial cells generated from pluripotent stem cells.     

低频电磁场在基因修饰表皮干细胞移植修复创面中的作用

背景：烧伤、慢性创面等大面积皮肤缺损的创面修复是临床上亟待解决的难题。表皮干细胞和角质化细胞生长因子可为创面的治疗提供新的策略。低频电磁场作为一种非侵入性物理刺激在创面修复中已被认为较佳的方法。目的：探讨低频电磁场在角质化细胞生长因子基因修饰的表皮干细胞移植促进小鼠皮肤全层缺损创面修复中的作用。方法：体外分离培养SD乳鼠表皮干细胞,用5-溴脱氧尿核苷进行标记,成功转染角质化细胞生长因子基因腺病毒表达载体后,移植于昆明小鼠全层缺损创面。建立全层缺损创面小鼠模型,分别进行表皮干细胞移植,角质化细胞生长因子修饰的表皮干细胞移植,角质化细胞生长因子修饰的表皮干细胞移植外加低频电磁场干预。结果与结论：移植后第9,16天,低频电磁场干预的角质化细胞生长因子基因修饰的表皮干细胞移植组创面收缩率优于其他各组（P〈0.05）。移植后第9天,免疫荧光染色法检测显示,各组组创面中均有5-溴脱氧尿核苷阳性细胞分布。移植后第16天,Westernblot检测显示,低频电磁场干预的角质化细胞生长因子基因修饰的表皮干细胞移植组和角质化细胞生长因子基因修饰的表皮干细胞移植组创面组织中角质化细胞生长因子蛋白表达高于表皮干细胞移植组（P〈0.05）;移植后第16,30天,苏木精-伊红染色结果显示,低频电磁场干预的角质化细胞生长因子基因修饰的表皮干细胞移植组的创面组织上皮化现象较明显。结果证实,低频电磁场有促进角质化细胞生长因子基因修饰的表皮干细胞移植修复小鼠皮肤全层缺损创面的作用。     

表皮干细胞联合脱细胞真皮构建人工皮肤促进创面愈合的实验研究

目的探讨表皮干细胞联合脱细胞真皮构建人工皮肤促进创面愈合的可行性。方法（1）用胰蛋白酶和EDTA联合消化法分离表皮,用Ⅳ型胶原快速黏附法分离、纯化人表皮干细胞,以含表皮生长因子、角质细胞无血清培养液等组成人表皮干细胞培养基进行体外培养,测定克隆形成率。（2）将培养人表皮干细胞接种于制备的脱细胞真皮支架中,构建组织工程人工皮肤,移植治疗兔全层皮肤缺损创面,观察创面修复效果。（3）取新西兰白兔常规制作背部全层皮肤缺损创面,随机分为4组：A、B、C组分别用含表皮干细胞的组织工程皮肤、含角质细胞的组织工程皮肤和单纯脱细胞真皮移植于皮肤缺损创面;D组用创面空置为对照。观察创面修复情况、局部炎症反应,并记录创面愈合时间。结果体外培养的人表皮干细胞增殖稳定,克隆形成率明显高于角质细胞对照组（P〈0．05）。移植后A组创面愈合良好,局部炎症反应轻微,无出血、积脓、坏死,创面愈合时间较B、C、D组明显缩短（P〈0．05或P〈0．01）。结论以表皮干细胞作为种子细胞联合脱细胞真皮构建人工皮肤可用于皮肤缺损创面的修复治疗。     

糖尿病模型皮肤创面中人真皮多能干细胞向表皮细胞的分化

背景：研究表明,干细胞分化为何种细胞与其所处的微环境密切相关。因此设想：人真皮多能干细胞处于皮肤创面的微环境中,可能具有向人皮肤细胞转化的潜能。目的：探讨糖尿病皮肤创面微环境中的人真皮多能干细胞分化为表皮细胞的可能性。方法：分离培养人真皮多能干细胞并制作糖尿病裸鼠模型皮肤创面,将标记5-BrdU的第3~5代人真皮多能干细胞以注射方式回植入糖尿病裸鼠创面组织周围,分别于注射后2,3周取材,常规石蜡包埋、连续切片,行BrdU和角蛋白免疫组织化学染色。结果与结论：BrdU阳性细胞出现在表皮中,且连续切片中部分BrdU阳性细胞也同时表达角蛋白。说明在糖尿病创面愈合过程中,人真皮多能干细胞具有向表皮细胞分化的潜能。     

Explant culture: a simple,reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate

Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue‐derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time‐consuming process. Mechanical stress during isolation, use of bacterial‐derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose explant culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by explant culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the explant‐derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As explant culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue‐derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material. Copyright © 2012 John Wiley & Sons, Ltd.     

Pluripotency rush! Molecular cues for pluripotency, genetic reprogramming of adult stem cells, and widely multipotent adult cells

In the last few years, pluripotent stem cells have been the objective of intense investigation efforts. These cells are of paramount therapeutic interest, since they could be utilized: as in vitro models of disease, for pharmaceutical screening purposes, and for the regeneration of damaged organs. Over the years, pluripotent cells have been cultured from teratomas, the inner cell mass, and primordial germ cells. Accumulating informations have partially decrypted the molecular machinery responsible for the maintenance of a very primitive state, permitting the reprogramming of differentiated cells.Although the debate is still open, an extreme excitement is arising from two strictly related possibilities: pluripotent cells could be obtained from adult tissues with minimal manipulations or very rare pluripotent cells could be identified in adult tissues. This intriguing option will trigger new researches aimed both at identifying the possible biological role of pluripotent adult stem cells and at exploiting their potential clinical use. The present review article will summarize current knowledge of the molecular cues for pluripotency but also discusses whether pluripotent stem cells could be obtained from adult tissues.     

Comparison of in vitro‐cultivation of human mesenchymal stroma/stem cells derived from bone marrow and umbilical cord

Cell‐mediated therapy is currently considered as a novel approach for many human diseases. Potential uses range from topic applications with the regeneration of confined tissue areas to systemic applications. Stem cells including mesenchymal stroma/stem cells (MSCs) represent a highly attractive option. Their potential to cure or alleviate human diseases is investigated in a number of clinical trials. A wide variety of methods has been established in the past years for isolation, cultivation and characterization of human MSCs as expansion is presently deemed a prerequisite for clinical application with high numbers of cells carrying reproducible properties. MSCs have been retrieved from various tissues and used in a multitude of settings whereby numerous experimental protocols are available for expansion of MSCs in vitro. Accordingly, different isolation, culture and upscaling techniques contribute to the heterogeneity of MSC characteristics and the, sometimes, controversial results. Therefore, this review discusses and summarizes certain experimental conditions for MSC in vitro culture focusing on adult bone marrow‐derived and neonatal umbilical cord‐derived MSCs in order to enhance our understanding for MSC tissue sources and to stratify different procedures. Copyright © 2016 John Wiley & Sons, Ltd.     

Human adipose tissue‐derived tenomodulin positive subpopulation of stem cells: A promising source of tendon progenitor cells

Cell‐based therapies are of particular interest for tendon and ligament regeneration given the low regenerative potential of these tissues. Adipose tissue is an abundant source of stem cells, which may be employed for the healing of tendon lesions. However, human adult multipotent adipose‐derived stem cells (hASCs) isolated from the stromal vascular fraction of adipose tissue originate highly heterogeneous cell populations that hinder their use in specific tissue‐oriented applications. In this study, distinct subpopulations of hASCs were immunomagnetic separated and their tenogenic differentiation capacity evaluated in the presence of several growth factors (GFs), namely endothelial GF, basic‐fibroblast GF, transforming GF‐β1 and platelet‐derived GF‐BB, which are well‐known regulators of tendon development, growth and healing. Among the screened hASCs subpopulations, tenomodulin‐positive cells were shown to be more promising for tenogenic applications and therefore this subpopulation was further studied, assessing tendon‐related markers (scleraxis, tenomodulin, tenascin C and decorin) both at gene and protein level. Additionally, the ability for depositing collagen type I and III forming extracellular matrix structures were weekly assessed up to 28 days. The results obtained indicated that tenomodulin‐positive cells exhibit phenotypical features of tendon progenitor cells and can be biochemically induced towards tenogenic lineage, demonstrating that this subset of hASCs can provide a reliable source of progenitor cells for therapies targeting tendon regeneration.     

人皮肤真皮组织中角朊干细胞样细胞的分离与培养

背景：皮肤真皮组织是皮肤损伤修复时除表皮基底层以外可形成表皮层结构的重要干细胞来源。目的：探索从人皮肤真皮组织中分离培养角朊干细胞样细胞的新方法。方法：将一定大小的成人腹部全层皮肤组织用Dispase过夜消化后去除表皮层结构,然后用0．1％I型胶原酶过夜消化,离心分离以获取真皮来源总细胞。将分离的细胞用含有体积分数1％N2,2％B27,20μg／L表皮生长因子和40pg／L碱性成纤维细胞生长因子的DMEM／F12（1：1）无血清培养液悬浮后以低密度接种于培养皿进行培养。采用RT-PCR和免疫细胞荧光染色法对其进行分析。结果与结论：RT-PCR分析结果显示所分离的真皮细胞中含有表达Lgr5和Lirgl等特异基因的毛囊来源干细胞;免疫细胞荧光分析结果显示其表达细胞角蛋白8和细胞角蛋白14。在无血清条件下培养6d时,可观察到表达细胞角蛋白14和整合素α6的“铺路石”样细胞克隆。进行传代培养后P1细胞表达角朊干细胞标志物CD29、细胞角蛋白14和P63。传代培养至P3,4时大部分细胞分化,不能继续传代。结果证实,以无血清培养液结合低密度培养法可直接从人真皮组织中有效地培养增殖活跃、细胞角蛋白14和P63阳性的角朊干细胞样细胞。     

大鼠骨髓间充质干细胞重建表皮细胞及皮肤附件简

背景：研究报道用烧伤大鼠血清作为诱导剂可以诱导间充质干细胞分化为表皮细胞。目的：体外诱导骨髓间充质干细胞分化为表皮细胞,单独或与必要的诱导剂组合移植修复皮肤创面缺损与重建表皮。方法：无菌环境取大鼠骨髓,选取贴壁细胞用L-DMEM培养液培养至第4代,通过流式细胞术鉴定骨髓间充质干细胞。用含20%烧伤大鼠血清 DMEM-F12培养液诱导骨髓间充质干细胞分化成表皮细胞,通过免疫组织化学鉴定。将Wistar大鼠制造全层皮肤缺损创面,分为3组,将BrdU标记的自体骨髓间充质干细胞及含有经诱导的骨髓间充质干细胞分别单次涂布到烧伤大鼠模型创面上,以未移植骨髓间充质干细胞做对照,观察再生皮肤创面收缩率及表皮层细胞与皮肤附件再生情况。结果与结论：分离、培养的骨髓间充质干细胞24 h后少部分细胞贴壁生长,形态长梭形,类似成纤维细胞,16 d时细胞贴满瓶底,呈鱼群样或网状排列,经流式细胞仪鉴定后加入20%烧伤大鼠血清的DMEM F-12培养基继续培养,细胞形态渐变为圆形或椭圆形细胞,经免疫组织细胞化学法和流式细胞术分析细胞角蛋白表达阳性,证实已分化为表皮细胞。动物实验结果表明无论是骨髓间充质干细胞单独移植还是与必要的诱导剂组合移植其皮肤的修复与再生情况均超过大鼠皮肤自然愈合,且不仅皮肤再生快,而且皮肤附件如毛囊等的再生也比对照组明显改善。初步推断间充质干细胞参与了表皮和皮肤附件毛囊的重建,从而改善皮肤愈合方式。     

Chemoattractant activity of degradation products of fetal and adult skin extracellular matrix for keratinocyte progenitor cells

Biological scaffolds composed of naturally occurring extracellular matrix (ECM) have been utilized as templates for the constructive remodelling of numerous tissues in preclinical studies and human clinical applications. The mechanisms by which ECM induces constructive remodelling are not well understood, but it appears that the degradation products of ECM scaffolds may play key roles in cell recruitment. The objective of the present study was to investigate the effects of age and species of the tissue from which ECM is harvested on the chemoattractant activity of degradation products of ECM for human keratinocyte stem and progenitor cells. Adult human skin ECM, fetal human skin ECM and adult porcine skin ECM were prepared, enzymatically digested, characterized by SDS-PAGE and evaluated for in vitro chemoattractant activity for human keratinocyte progenitor and stem cells (HEKn). Degradation products of human fetal skin ECM showed greater chemoattractant activity than human adult skin ECM degradation products for the HEKn. Degradation products of porcine adult skin ECM showed greater chemoattractant activity than human adult skin ECM. The human fetal skin ECM degradation products showed the strongest chemoattractant activity for the HEKn. The findings of this study support the concept that the mechanism of ECM scaffold remodelling involves the recruitment of lineage-directed progenitor cells by scaffold degradation products, and that both the age and species of the tissue from which the ECM is harvested have an effect upon this chemoattractant potential.     

Adult mesenchymal stromal stem cells for therapeutic applications

Mesenchymal stromal stem cells (MSC) can be found in almost any adult organ. They can be isolated and expanded within several weeks up to hundreds of millions of cells. The cell isolation based on the surface antigen expression may significantly enrich for the desired cell population and reduce the time required for cell expansion. MSC display a unique molecular signature which clearly discriminates them from other stem cell types. MSC can be differentiated into the cells of several lineages. Additionally, the unique biological properties of MSC are mediated by strong immunomodulatory activity and by paracrine mechanisms. Potential therapeutic applications of the cells require clinically compliant protocols for cell isolation and expansion. The therapeutic utility of MSC has been evaluated and found to be useful in several pre‐clinical animal models as well as in clinical trials.     

Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells

Xeroderma pigmentosum (XP) is a devastating disease associated with dramatic skin cancer proneness. XP cells are deficient in nucleotide excision repair (NER) of bulky DNA adducts including ultraviolet (UV)-induced mutagenic lesions. Approaches of corrective gene transfer in NER-deficient keratinocyte stem cells hold great hope for the long-term treatment of XP patients. To face this challenge, we developed a retrovirus-based strategy to safely transduce the wild-type XPC gene into clonogenic human primary XP-C keratinocytes. De novo expression of XPC was maintained in both mass population and derived independent candidate stem cells (holoclones) after more than 130 population doublings (PD) in culture upon serial propagation (>10(40) cells). Analyses of retrovirus integration sequences in isolated keratinocyte stem cells suggested the absence of adverse effects such as oncogenic activation or clonal expansion. Furthermore, corrected XP-C keratinocytes exhibited full NER capacity as well as normal features of epidermal differentiation in both organotypic skin cultures and in a preclinical murine model of human skin regeneration in vivo. The achievement of a long-term genetic correction of XP-C epidermal stem cells constitutes the first preclinical model of ex vivo gene therapy for XP-C patients.     

昆明种小鼠胚胎神经干细胞的分离及培养

目的：在神经干细胞的分离培养过程中，国内外的报道多数是用胰蛋白酶对组织细胞进行消化，但是消化时间比较难把握。采用胰酶消化与机械分离法相结合的方式对昆明种小鼠胚胎脑神经干细胞进行分离、培养和初步的免疫组织化学检测，为相关研究建立实验条件。 方法：实验于2006—10／2007-09在广西医科大学基础医学实验室完成。实验室为广西重点实验室、基础医学博士后工作站。①实验材料：孕14-16d的昆明种小鼠由广西医科大学实验动物中心提供，实验过程中对动物处置符合动物伦理学标准。②实验方法：分离昆明种小鼠胎鼠的脑组织，经胰酶消化加机械吹打后，在加入碱性成纤维细胞生长因子和表皮细胞生长因子的B27无血清培养基中培养。③实验评估：用免疫细胞化学方法鉴定分离的神经干细胞。 结果：在无血清DMEM／F12培养液中，加入碱性成纤维细胞生长因子、表皮细胞生长因子及B27的条件下，培养24h后可见细胞以悬浮方式生长，三五聚集成团块，48h后形成由十数个至数十个细胞组成的，大小不等的细胞球，形态规则，细胞无突起，形成典型的神经球，可传代。免疫细胞化学法检测显示，细胞表达神经巢蛋白。 结论：①来自昆明种小鼠胎脑的神经干细胞能在体外培养、增殖并具有增殖传代能力。②无血清B27培养基添加表皮细胞生长因子、碱性成纤维细胞生长因子有利于神经干细胞的体外培养和促进其增殖。     

昆明种小鼠胚胎神经干细胞的分离与培养

背景：对于神经千细胞的分离培养,目前多采用胰蛋白酶对组织细胞予以消化,但消化时间较难把握。 目的：采用胰酶消化与机械分离法相结合的方式对昆明种小鼠胚胎脑神经干细胞进行分离、培养,并进行初步的免疫组织化学检测。 设计、时间及地点：细胞学体外观察,于2006～10/2007—09在广西医科大学基础医学实验室完成。材料：孕14-16d的昆明种小鼠由广西医科大学实验动物中心提供。 方法：分离昆明种小鼠胎鼠的脑组织,经胰酶消化加机械吹打后,在加入碱性成纤维细胞生长因子和表皮细胞生长因子的B27无血清DMEMIF12培养基中培养。主要观察指标：用免疫细胞化学方法鉴定分离的神经干细胞。结果：培养24h后,细胞以悬浮方式生长,聚集成团：48h后形成由数十个细胞组成的细胞球,形态规则,体积大小不等,细胞无突起,形成典型的神经球,可传代扩增。免疫细胞化学染色结果示细胞巢蛋白呈阳性表达。 结论：在含有表皮细胞生长因子、碱性成纤维细胞生长因子的无血清B27培养基条件下,有利于小鼠胚胎神经干细胞的体外培养和传代增殖。     

Simvastatin induces the osteogenic differentiation of human periodontal ligament stem cells

Periodontal ligament stem cells (PDLSCs) are considered as potential mesenchymal stem cell sources for future clinical applications in periodontal regeneration therapy. Simvastation, widely used for lowering serum cholesterol, is known to have a bone stimulatory effect. However, it is not clear whether simvastation affects the differentiation of PDLSCs. This study examined the effects of simvastatin on human PDLSCs in vitro and in vivo. Using the limiting dilution technique, human PDLSCs were isolated and expanded. PDLSCs were cultured with simvastatin (0.01–10 μm ), and the proliferation was measured. The osteogenic differentiation was characterized by alkaline phosphatase (ALP) activity and Alizarin Red‐S staining for calcium deposition. The gene expression levels of osteogenic markers were evaluated by RT‐PCR. In addition, PDLSCs were transplanted into nude mice with ceramic bovine bone powders as carriers to observe the capacity of mineralized tissue formation in vivo. Simvastatin at concentrations <1 μm did not suppress the proliferation of PDLSCs. After the administration of 0.1 μm simvastatin, the expression of ALP, bone sialoprotein, and bone morphogenetic protein‐2 genes were significantly upregulated, and the ALP activity and mineralized nodule formation were significantly higher in the simvastatin‐treated cells than the control cells. In addition, the in vivo transplantation results showed that simvastatin treatment promoted the degree of mineralized tissue formation. Collectively, simvastatin has positive effects on osteogenic differentiation of human PDLSCs in vitro and in vivo. This suggests that simvastatin might be a useful osteogenic induction agent for periodontal bone regeneration.     

Mesenchymal stem cells

About 40 years ago Friedenstein described stromal cells in the bone marrow that were spindle shaped and proliferate to form colonies. These cells attach to plastic and are able to differentiate under defined in vitro conditions into multiple cell types present in many different tissues, e.g. osteoblasts, chondroblasts, adipocytes, etc. Later on these cells, obtained from postnatal bone marrow, were called mesenchymal stem cells (MSC) or stromal stem cells. Recently the presence of somewhat similar cells has been demonstrated in many other tissues too. In spite of extensive attempts to characterize these cells we are still lacking definitive in vivo markers of MSC although retrospective functional data strongly support the existence of common adult stem cells that have the capacity to differentiate along various specific differentiation lineages. Since MSC can be rather easily isolated from the bone marrow and can also be expanded in vitro they have become a prime target for researchers of tissue regeneration. These cells have now been extensively used for transplantation experiments in animals and also for some therapeutic trials in humans. However, much new research is needed to learn enough on the molecular mechanisms of MSC differentiation to evaluate their full capacity for tissue regeneration.     

Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic‐like differentiation

Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen‐stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well‐differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic‐like differentiation with morphological change from a spindle‐shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver‐specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation‐medium culture. Positive immunofluorescence staining of low‐density lipoprotein and albumin was observed from day 14 of differentiation‐medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic‐like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic‐like cells. Copyright © 2013 John Wiley & Sons, Ltd.