Identification and Isolation of Human Dental Pulp Stem Cells

1 Introduction Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts,that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue.     

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson’s disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3–4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6–8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10–15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson’s disease.     

Multipotent Differentiation of Human Dental Pulp Stem Cells: a Literature Review

The advent of regenerative medicine has brought us the opportunity to regenerate, modify and restore human organs function. Stem cells, a key resource in regenerative medicine, are defined as clonogenic, self-renewing, progenitor cells that can generate into one or more specialized cell types. Stem cells have been classified into three main groups: embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult/postnatal stem cells (ASCs). The present review focused the attention on ASCs, which have been identified in many perioral tissues such as dental pulp, periodontal ligament, follicle, gingival, alveolar bone and papilla. Human dental pulp stem cells (hDPSCs) are ectodermal-derived stem cells, originating from migrating neural crest cells and possess mesenchymal stem cell properties. During last decade, hDPSCs have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and ability to differentiate in several cell phenotypes. In this review, we have carefully described the potential of hDPSCs to differentiate into odontoblasts, osteocytes/osteoblasts, adipocytes, chondrocytes and neural cells.     

A Journey from Dental Pulp Stem Cells to a Bio-tooth

The ultimate goal of tooth regeneration is to replace the lost teeth. Stem cell-based tooth engineering is deemed as a promising approach to the making of a biological tooth (bio-tooth). Dental pulp stem cells (DPSCs) represent a kind of adult cell colony which has the potent capacity of self-renewing and multilineage differentiation. The exact origin of DPSCs has not been fully determined and these stem cells seem to be the source of odontoblasts that contribute to the formation of dentin-pulp complex. Recently, achievements obtained from stem cell biology and tooth regeneration have enabled us to contemplate the potential applications of DPSCs. Some studies have proved that DPSCs are capable of producing dental tissues in vivo including dentin, pulp, and crown-like structures. Whereas other investigations have shown that these stem cells can bring about the formation of bone-like tissues. Theoretically, a bio-tooth made from autogenous DPSCs should be the best choice for clinical tooth reconstruction. This review will focus on the location, origin, and current isolation approaches of these stem cells. Their odontoblastic differentiation and potential utilizations in the reconstruction of dentin-pulp complex and bio-tooth will be extensively discussed.     

人胚脑皮层神经干细胞的分离培养及鉴定

目的 从人胚脑皮层中分离培养并鉴定神经干细胞，方法 利用无血清培养和单细胞克隆技术在人胚脑皮层中分离出具有单细胞克隆能力的细胞，并进行培养、传代、分化观察，采用间接免疫荧光检测克隆细胞的神经巢蛋白（Nestin)抗原和分化后特异性成熟神经细胞抗原的表达。结果 从胚龄10周的新鲜人胚脑皮层中成功分离出神经干细胞，该细胞具有连续克隆能力，可传代培养，表达神经巢蛋白抗原，分化后的细胞表达神经元细胞，胶质细胞和少突胶质细胞的特异性抗原。结论 人胚脑皮层中存在具有自我更新能力和多分化潜能的神经干细胞。     

Dental Pulp Stem Cells: A Promising Tool for Bone Regeneration

Human tissues are different in term of regenerative properties. Stem cells are a promising tool for tissue regeneration, thanks to their particular characteristics of proliferation, differentiation and plasticity. Several “loci” or “niches” within the adult human body are colonized by a significant number of stem cells. However, access to these potential collection sites often is a limiting point. The interaction with biomaterials is a further point that needs to be considered for the therapeutic use of stem cells. Dental pulp stem cells (DPSCs) have been demonstrated to answer all of these issues: access to the collection site of these cells is easy and produces very low morbidity; extraction of stem cells from pulp tissue is highly efficiency; they have an extensive differentiation ability; and the demonstrated interactivity with biomaterials makes them ideal for tissue reconstruction. SBP-DPSCs are a multipotent stem cell subpopulation of DPSCs which are able to differentiate into osteoblasts, synthesizing 3D woven bone tissue chips in vitro and that are capable to synergically differentiate into osteoblasts and endotheliocytes. Several studied have been performed on DPSCs and they mainly found that these cells are multipotent stromal cells that can be safety cryopreserved, used with several scaffolds, that can extensively proliferate, have a long lifespan and build in vivo an adult bone with Havers channels and an appropriate vascularization. A definitive proof of their ability to produce dentin has not been yet done. Interestingly, they seem to possess immunoprivileges as they can be grafted into allogenic tissues and seem to exert anti-inflammatory abilities, like many other mesenchymal stem cells. The easy management of dental pulp stem cells make them feasible for use in clinical trials on human patients. An erratum to this article can be found at     

大鼠肝卵圆细胞的诱导、分离及鉴定

目的建立大鼠肝卵圆细胞的增殖模型,并探索其分离及鉴定方法。方法雄性Wistar大鼠每天1次连续灌胃给予不同剂量二乙酰氨基芴(2-AAF熏5、10、15、20、25mg/kgBW),第5天行标准的2/3肝切除术,术后按各自剂量继续给予11天,不同时间取肝脏组织,行甲胎蛋白、细胞角蛋白18及19染色并观察。以确定的2-AAF最佳剂量制备大鼠肝干细胞增殖模型,Seglen胶原酶原位灌注结合Percoll密度梯度离心分离纯化大鼠肝卵圆细胞,光镜、电镜下观察细胞特点,并进行上述细胞表型标志免疫组化染色。结果2-AAF15mg/kgBW能建立较理想的肝卵圆细胞增殖模型。HE染色可见汇管区及中央静脉周围大量增殖的嗜碱性小细胞,电镜下观察此种细胞具有卵圆形细胞核、细胞质少而淡、核/浆比例较大等特点,免疫组化染色证实甲胎蛋白、细胞角蛋白18和19染色阳性,白蛋白及白细胞共同抗原(LCA)染色阴性。分离所得底层细胞,光镜下表现大小不等、不规则圆形细胞,体积较小,细胞核/浆比例较大,电镜下细胞表面可见少量短而小的微绒毛状突起,余同增殖细胞特点,免疫组化染色与增殖细胞表现相同细胞表型特点。结论本方法可成功诱导、分离、纯化大鼠肝卵圆细胞,符合肝卵圆细胞的形态特点、超微结构及细胞表型标志特点。     

大鼠牙髓细胞的分离培养及超微结构观察

目的 探讨牙髓细胞的培养方法和生长状况,观察其形态结构特征,为进一步研究牙齿的发育提供实验基础.方法 采用胶原酶消化分离的方法分离大鼠的牙髓细胞并在DMEM培养液中培养,运用透射和扫描电镜技术观察细胞的形态结构特征.结果 培养的牙髓细胞呈梭形,细胞生长状况良好,细胞器发育正常,并且有细胞增殖和分裂能力.结论 利用酶消化分离的方法分离培养的牙髓细胞,能够保持其生活状态及增殖分裂能力.     

Dental Pulp Tissue Engineering Using Mesenchymal Stem Cells: a Review with a Protocol

Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from human and animal sources such as rats. Recently, an in vivo protocol for pulp tissue engineering using implantation of bone marrow MSCs into rat pulpotomized molars was established by our research group. This coronal pulp regeneration model showed almost complete regeneration/healing with dentin bridge formation when the cavity was sealed with mineral trioxide aggregate (MTA) to create a biocompatible seal of the pulp. This method is a powerful tool for elucidating the processes of dental pulp tissue regeneration following implantation of MSCs. In the present review, we discuss the literature in the field of dental pulp tissue engineering using MSCs including dental pulp stem cells and stem cells from exfoliated deciduous teeth. In addition, we present a brief step-by-step protocol of the coronal pulp regeneration model focusing on the implantation of rat bone marrow MSCs, biodegradable scaffolds, and hydrogels in pulpotomized rat molars. The protocol may lay the foundation for studies aiming at defining further histological and molecular mechanism of the rat pulp tissue engineering.     

Human Neural Stem Cells: Isolation, Expansion and Transplantation

Neural stem cells, with the capacity to self renew and produce the major cell types of the brain, exist in the developing and adult rodent central nervous system (CNS). Their exact function and distribution is currently being assessed, but they represent an interesting cell population, which may be used to study factors important for the differentiation of neurons, astrocytes and oligodendrocytes. Recent evidence suggests that neural stem cells may also exist in both the developing and adult human CNS. These cells can be grown in vitro for long periods of time while retaining the potential to differentiate into nervous tissue. Significantly, many neurons can be produced from a limited number of starting cells, raising the possibility of cell replacement therapy for a wide range of neurological disorders. This review summarises this fascinating and growing field of neurobiology, with a particular focus on human tissues.     

Human Dental Pulp-Derived Stem Cells Restore Movement in Rats with Spinal Cord Injuries

Spinal cord injuries (SCIs) are a prevailing problem worldwide, with over 250,000 Americans cur- rently affected in which the individual is rendered either paraplegic     

Pluripotent Human Stem Cells

The need for new and improved pharmacotherapies in medicine, high late-stage compound attrition in drug discovery, and upcoming patent expirations is driving interest by the pharmaceutical industry in pluripotent stem cells for in vitro modeling and early-stage testing of toxicity and target engagement. In particular, human embryonic and induced pluripotent stem cells represent potentially cost-effective and accessible sources of organ-specific cells that foretell in vivo human tissue response to new chemical entities. Here we consider the potential of these cells as novel tools for drug development, including toxicity screening and metabolic profiling. We hold that despite various challenges to translating proof-of-concept screening platforms to industrial use, the promise of research is considerable, and close to being realized.     

Effective Differentiation of Induced Pluripotent Stem Cells Into Dental Cells

Background: A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell-based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. Results: Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. Conclusions: Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129–139, 2019. © 2018 Wiley Periodicals, Inc.     

人胚神经干细胞的分离、培养与鉴定

为了探讨人胚神经干细胞体外培养条件和分化情况，摸索出一种切实可行的获得较纯、多潜能人 胚神经干细胞的方法。我们取三月龄人胎脑，用胰蛋白酶消化法分离单个细胞，部分冻存，另一部分进行细胞培养，加EGF、bFGF刺激生长，有限稀释法将获得单细胞克隆，血清诱导分化，并用免疫组化方法进行鉴定。结果显示,EGF和bFGF同时存在的无血清培养基中有大量神经干细胞团生成，含血清培养基则诱导神经干细胞分化成为神经元、星型胶质细胞、少 突胶质细胞。这表明，神经干细胞的存活和分裂有赖于EGF和bEGF的共同作用。经冻存后的胎脑细胞同样能分离培养出有活性的神经干细胞。     

胎盘来源的间充质干细胞体外分离培养及向软骨细胞分化的研究

目的对人胎盘间充质干细胞体外分离培养及向软骨细胞分化进行研究,为软骨组织工程提供种子细胞。方法取人正常分娩后的胎盘,用组织块培养法分离获取胎盘间充质干细胞,对所获细胞进行体外培养和表面标志的流式细胞仪分析。对经过证实的骨髓间充质干细胞用含TGF-β_3、维生素C、胰岛素、地塞米松、转铁蛋白的诱导培养基处理7-28d,对诱导细胞进行甲苯胺蓝染色及Ⅱ型胶原染色,鉴定诱导后的软骨细胞表型。结果培养的人胎盘间充质干细胞均一表达CD_(29)和CD_(44),而CD_(31)、CD_(34)、CD_(45)和HLA-DR呈阴性。细胞经诱导液作用14d后,甲苯胺蓝染色及Ⅱ型胶原染色阳性。结论采用组织块培养法能分离获取高纯度的人胎盘间充质干细胞,该细胞经诱导液作用可定向分化为软骨细胞。     

Induced Pluripotent Stem Cells from Human Hair Follicle Mesenchymal Stem Cells

Reprogramming of somatic cells into inducible pluripotent stem cells (iPSCs) provides an alternative to using embryonic stem cells (ESCs). Mesenchymal stem cells derived from human hair follicles (hHF-MSCs) are easily accessible, reproducible by direct plucking of human hairs. Whether these hHF-MSCs can be reprogrammed has not been previously reported. Here we report the generation of iPSCs from hHF-MSCs obtained by plucking several hairs. hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature confirmed by detecting cell surface antigens and multilineage differentiation potential towards adipocytes and osteoblasts. They were then reprogrammed into iPSCs by lentiviral transduction with Oct4, Sox2, c-Myc and Klf4. hHF-MSC-derived iPSCs appeared indistinguishable from human embryonic stem cells (hESCs) in colony morphology, expression of alkaline phosphotase, and expression of specific hESCs surface markers, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, Nanog, Oct4, E-Cadherin and endogenous pluripotent genes. When injected into immunocompromised mice, hHF-MSC-derived iPSCs formed teratomas containing representatives of all three germ layers. This is the first study to report reprogramming of hHF-MSCs into iPSCs.     

Human Immature Dental Pulp Stem Cells (hIDPSCs), Their Application to Cell Therapy and Bioengineering: An Analysis by Systematic Revision of the Last Decade of Literature

During recent years, attention has been given to the potential of therapeutic approaches using stem cells obtained from dental pulp tissue. The aim of this study, therefore, was to give an overview of the papers produced during the last 10 years that have described the use of stem cells obtained from human deciduous teeth in cell therapy or bioengineering. The PubMed database was investigated from January 2002 until July 2011 and the papers published during this period were analyzed according to criteria previously established, using the methodology of systematic review. The measurements were done using “stem cell” as the primary keyword, and “human deciduous teeth dental pulp cell” and “human exfoliated deciduous teeth” as the secondary keywords. Four hundred and seventy‐five papers were found. The first screening resulted in 276 papers, from which 84 papers were selected. However, only 11 of them attained the aim proposed in our approach. There were few scientific studies related to direct therapeutic application using stem cells of human deciduous teeth and none of them had been applied to humans. However, the results indicated important and promising applications of the pulp stem‐cells in cell therapy and bioengineering as demonstrated by studies in animal models of muscular dystrophy, Parkison's disease, and lupus erythematosus. Anat Rec, 296:1923–1928, 2013. © 2013 Wiley Periodicals, Inc.