Modulation of stem cell shape and fate A: the role of density and seeding protocol on nucleus shape and gene expression

Mesenchymal stem cell shape and fate are intrinsic manifestations of form and function at the cellular level. We hypothesize that cell seeding density and initial seeding protocol influence stem cell shape and fate. Nucleus shape and early (within days of seeding) expression of genes typical for pre-, peri-, and postcondensation events were compared between groups of cells after seeding at or proliferating to target density (low density [LD], 16,500 cells/cm2; high density [HD], 35,000 cells/cm2; very high density [VHD], 86,500 cells/cm2). Significant differences in nuclear shape could be attributed to seeding protocol in the VHD group, where nuclei from cells that proliferated to VHD were significantly rounder than nuclei from cells seeded at target VHD. Furthermore, cells that proliferated to VHD exhibited significantly rounder nuclei than nuclei from all other cell density and seeding protocol groups. In contrast, nuclei from cells that were seeded at the VHD were flatter than nuclei from cells of all other groups. Furthermore, the significant rounding of nuclei in the cells that proliferated to VHD was accompanied by a two-, six-, and ninefold increase from baseline in Runx2, Sox9, and Aggrecan (AGC) expression, markers indicative of precondensation, peri-, and post-condensation events, respectively. None of the other groups showed significant changes in gene expression over baseline. Finally, seeding at target density results in greater overlap of cells compared to groups in which cells proliferate to target density, conferring increased thickness to multicellular culture aggregates seeded at target density. These data suggest that seeding protocols can be exploited to modulate mesenchymal stem cell shape and early gene expression typical for condensation events in development, which occur over an approximately 12-h period at E11.5 in the mouse limb bud. Follow-on studies will delineate longer-term effects of density and seeding protocol on modulation of stem cell fate and cell assembly to form tissues.     

Bone morphogenetic protein rescues the lack of secondary cartilage in Runx2-deficient mice

Secondary cartilages including mandibular condylar cartilage have unique characteristics. They originate from alkaline phosphatase (ALP)-positive progenitor cells of the periosteum, and exhibit characteristic modes of differentiation. They also have a unique extracellular matrix, and coexpress type I, II and X collagens. We have previously shown that there is a total absence of secondary cartilages in Runx2-deficient (Runx2-/-) mice. To clarify whether Runx2 is essential for chondrocytic differentiation of secondary cartilages, we performed an organ culture system using mandibular explants derived from Runx2-/- mice at embryonic day 18.0. Since mRNA for bone morphogenetic protein 2 (BMP2) was strongly expressed in osteoblasts of condylar anlagen in wild-type mice, and was down-regulated in those of Runx2-/- mice, we chose to investigate BMP2 effects on secondary cartilage formation. Condensed mesenchymal cells of mandibular condylar anlagen in precultured explants were ALP-positive and expressed type I collagen and Sox9. After culture with recombinant human (rh) BMP2, chondrocytic cells showing ALP activity and expressing Sox5, Sox9, and type I and II collagens, appeared from mesenchymal condensation. This expression profile was comparable with the reported pattern of chondrocytes in mouse secondary cartilages. However, chondrocyte hypertrophy was not observed in the explants. These findings indicate that BMP2 partially rescued chondrocyte differentiation but not chondrocyte hypertrophy in secondary cartilage formation in Runx2-/- mice. Runx2 is required for chondrocyte hypertrophy in secondary cartilage formation, and it is likely that BMP2, which is abundantly secreted by osteoblasts in condylar anlagen, contributes to the early process of secondary cartilage formation.     

miR-155有助于骨髓间充质干细胞成软骨分化

背景：近年发现,miRNA是能够对基因表达产生影响的一种新型调控子,miRNA有助于多能干细胞分化增殖以及自我更新。 目的：探讨miR-155对大鼠骨髓间充质干细胞成软骨分化的调控机制。 方法：12周龄健康SD大鼠60只,随机分为研究组与对照组,每组30只。于麻醉状态下将大鼠处死,获取下肢骨髓,进行骨髓间充质干细胞分离、培养,研究组给予miR-155基因模拟物转染,对照组给予阴性对照序列转染,经成软骨诱导分化后进行RT-PCR检测Sox9、CollagenⅡ、Aggrecan、Collagen X基因的表达,Western blot检测Sox9、Runx2蛋白的表达。 结果与结论：研究组Sox9、CollagenⅡ、Aggrecan基因表达高于对照组,Collagen X基因表达低于对照组,差异均有显著性意义(P < 0.05)。研究组Sox9蛋白表达高于对照组,Runx2蛋白表达低于对照组,差异均有显著性意义(P < 0.05)。结果表明miR-155不仅有助于骨髓间充质干细胞成软骨分化,而且可以抑制骨髓间充质干细胞成软骨分化呈肥大趋势发展。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

人脐带间充质干细胞表达胚胎干细胞相关转录因子

背景：Nanog、Oct4和Sox2通过调节胚胎干细胞的基因转录,对其多潜能性和自我更新的能力具有关键性的调控作用,脐带间充质干细胞中这些胚胎干细胞相关转录因子的表达情况如何还不太清楚。 目的：研究脐带间充质干细胞中Nanog、Oct4和Sox2等这些胚胎干细胞相关转录因子的表达情况。 方法：胶原酶和胰酶消化法培养脐带间充质干细胞;mTeSRTM1体系进行无滋养层培养人胚胎干细胞,定量PCR比较上述两种细胞中Nanog、Oct4和Sox2 mRNA表达量的差异;免疫荧光检测上述两种细胞中Nanog、Oct4和Sox2的表达情况。 结果与结论：间充质干细胞表达胚胎干细胞标记Nanog、Oct4和Sox2,但Oct4主要表达在胞浆,且以Oct4B为主。脐带间充质干细胞Nanog、Oct4A和Sox2的表达量明显低于胚胎干细胞,其mRNA表达量分别为胚胎干细胞的20%,0.3%,10%左右。通过了解两种细胞Nanog、Oct4和Sox2的表达差异,可为优化脐带间充质干细胞重编程提供依据,也为进一步研究胚胎干细胞相关转录因子在成体干细胞表达起何种作用提供参考。     

Up-regulated osteogenic transcription factors during early response of human periodontal ligament stem cells to cyclic tensile strain

Introduction

As one group of periodontal ligament (PDL) cells, human periodontal ligament stem cells (hPDLSCs) have been isolated and identified as mesenchymal adult stem cells (MSCs) since 2004. It has been well accepted that PDL sensitively mediates the transmission of stress stimuli to the alveolar bone for periodontal tissue remolding. Besides, the direction of MSCs differentiation has been verified regulated by mechanical signals. Therefore, we hypothesized that tensile strain might act on hPDLSCs differentiation, and the early response to mechanical stress should be investigated.

Material and methods

The hPDLSCs were cultured in vitro and isolated via a magnetic activated CD146 cell sorting system. After investigation of surface markers and other experiments for identification, hPDLSCs were subjected to cyclic tensile strain at 3,000 µstrain for 3 h, 6 h, 12 h, and 24 h, without addition of osteogenic supplements. In the control groups, the cells were cultured in similar conditions without mechanical stimulation. Then osteogenic related genes and proteins were analyzed by RT-PCR and western blot.

Results

Cyclic tensile strain at 3,000 µstrain of 6 h, 12 h, and 24 h durations significantly increased mRNA and protein expressions of Satb2, Runx2, and Osx, which were not affected in unloaded hPDLSCs.

Conclusions

We indicate that hPDLSCs might be sensitive to cyclic tensile strain. The significant increase of Runx2, Osx and Satb2 expressions may suggest an early response toward osteogenic orientation of hPDLSCs.     

果糖和二硫苏糖醇对骨髓间充质干细胞冻存后活性及多能性的影响

背景:冻存是保证干细胞临床应用的关键步骤之一,但现有冻存技术常导致细胞活性降低、多能性丧失及分化能力下降。目的:探究果糖及二硫苏糖醇是否有助于维持冻存后骨髓间充质干细胞多能性及成骨分化潜能。方法:分离培养SD大鼠骨髓间充质干细胞,在细胞冻存前分别用果糖(200μmol/L),二硫苏糖醇(500μmol/L)μmol/L)及果糖(200μmol/L)+二硫苏糖醇(500预处理1 h。冻存6个月后,复苏细胞并用倒置显微镜观察细胞形态,MTT实验检测细胞活性,定量PCR检测相关干性基因(Nanog,Oct4及Sox2)的表达,碱性磷酸酶活性测试及茜素红染色检测复苏骨髓间充质干细胞成骨分化能力。结果与结论:(1)复苏后各组细胞在形态上无明显差别;(2)果糖预处理及联合预处理有助于骨髓间充质干细胞活性维持;(3)二硫苏糖醇预处理可显著促进骨髓间充质干细胞多能性相关基因Nanog及Sox2的表达;(4)果糖、二硫苏糖醇及联合预处理皆有助于维持骨髓间充质干细胞成骨分化潜能,但以二硫苏糖醇及联合预处理组效果最佳;(5)结果表明,果糖预处理有助于维持冻存骨髓间充质干细胞活性,二硫苏糖醇有助于维持冻存骨髓间充质干细胞多能性及成骨分化能力。     

骨髓间充质干细胞Notch1信号通路异常与骨髓瘤骨病

背景：多发性骨髓瘤骨病的发病机制目前尚未完全明确,骨髓间充质干细胞向成骨细胞分化障碍参与其中,而Notch1信号通路在间充质干细胞的增殖分化中起重要作用。 目的：探讨Notch1信号通路在多发性骨髓瘤骨病中的作用。 方法：分离培养多发性骨髓瘤患者和正常人骨髓间充质干细胞,Real-time PCR和Western blot检测成骨诱导分化前后Notch1和成骨基因Runx2的表达,以及Von Kossa染色鉴定钙质沉积程度。在多发性骨髓瘤患者间充质干细胞成骨诱导分化过程中,加入Notch1信号通路抑制剂DAPT和安慰剂,48 h后real-time PCR和western blot鉴定Notch1信号通路下游分子Hes1和成骨指标Runx2表达,2周后Von Kossa染色鉴定钙质沉积程度。 结果与结论：成骨诱导48 h后,间充质干细胞的Notch1表达减低,但是骨髓瘤患者间充质干细胞的降低幅度小于正常对照间充质干细胞;48 h后Runx2的表达在骨髓瘤患者间充质干细胞的表达明显弱于正常对照间充质干细胞;2周后,Von Kossa染色鉴定钙质沉积程度,骨髓瘤患者间充质干细胞明显弱于正常对照间充质干细胞;48 h后Hes1表达在DAPT组明显低于安慰剂组;而Runx2的表达在DAPT组明显高于安慰剂组。2周后 DAPT组钙质沉积明显强于安慰剂组。实验说明多发性骨髓瘤患者的间充质干细胞中,Notch1信号通路失活缺陷可能抑制其向成骨细胞分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：