自体骨髓干细胞移植研究进展

细胞移植作为一种新的治疗方法备受医学界关注，但细胞供应短缺和免疫排斥是阻碍这一疗法临床推广的主要难题。自体骨髓干细胞移植在一定程度上可缓解此矛盾。文章综述了自体骨髓干细胞移植治疗脑、肝、心等重要器官疾病的基础和临床研究现状，以及骨髓动员剂在骨髓干细胞移植中的作用。     

人体骨髓基质干细胞冷冻干燥的探索性实验

人体骨髓基质干细胞已成为组织工程、细胞移植和基因治疗等领域的重要研究对象,目前对它已有低温保存的研究,而通过冷冻干燥的方法对其进行保存国内外未见报导.实验尝试用冻干的方法来保存骨髓基质干细胞.文中选取海藻糖、PVP、HES等作保护剂,应用差示扫描量热仪(DSC)测量其结晶温度和玻璃化转变温度;随后对加入保护剂的细胞溶液进行冻干实验,并应用流式细胞仪对冻干样品复水后的细胞活性进行了测定,其中30%PVP 20%海藻糖对细胞的保护效果较好,细胞成活率达到16.40%.     

骨髓4℃保存方法及移植治疗晚期实体瘤的研究

对小鼠和人骨髓用TC—199营养液、复方氨基酸加ATP和生理盐水在4℃的保存效果进行了比较研究。用国产复方氨基酸加ATP保存的自体骨髓,在晚期实体瘤患者行强化疗后输回自身,有效率为75％。该方法安全、有效,且经济、方便,适合于基层医院推广应用。     

大鼠骨髓间质干细胞的分离纯化与鉴定

采用密度梯度离心法分离骨髓间质干细胞,差速贴壁法进行纯化,应用流式细胞仪、激光共聚焦显微镜等对纯化细胞进行鉴定,结果细胞表面抗原CD２９,CD４４,CD１０５,CD１６６表达呈阳性,而CD１４,CD３４,CD４５表达呈阴性。采用RT唱PCR鉴定了三个基因：nestin,NST,Oct唱４,前两者呈阳性表达,后者弱阳性表达。这些细胞特异抗原与基因表达综合起来,表明得到的细胞具备骨髓间质干细胞的特性。密度梯度分离与差速贴壁相结合,可获得较好均一性的骨髓间质干细胞,是一种简单可靠、易于推广的骨髓干细胞获取方法,可为细胞与组织工程研究提供种子细胞。     

大鼠骨髓间充质干细胞与β-磷酸三钙/聚L-乳酸支架材料的复合

将成骨诱导前后的大鼠骨髓间充质干细胞与不同比例、不同孔径和不同孔隙率的βTCP／PLLA材料复合,通过体内体外实验研究β-磷酸三钙／聚L-乳酸（β-TCP／PLLA）的成分、孔径和孔隙率等结构参数对复合材料在体内异位成骨能力的影响．结果表明,β-TCP／PLLA成分比为2：1,致孔剂含量为70％;孔径为200-450μm的支架材料在体外更有利于细胞的生长、增殖以及分化．这种支架材料在裸鼠体内具有异位成骨的能力,成骨诱导后的大鼠骨髓间充质干细胞比没有诱导的细胞更适合作为种子细胞．     

体外壳聚糖-明胶-生长因子缓释支架对骨髓间充质干细胞增殖的影响

制备具有缓释功能的壳聚糖-明胶-碱性成纤维细胞生长因子(bFGF)-肝细胞生长因子(HGF)三维大孔支架,探讨两种生长因子在体外对骨髓间充质干细胞(BMSCs)增殖的协同作用。方法:采用冷冻干燥法,将不同比例的壳聚糖、明胶、bFGF和HGF依次混合,使其成为具有一定孔径的三维缓释支架。取SD大鼠乳鼠股骨和胫骨骨髓,分离、培养BMSCs。取生长状态良好的第三代BMSCs,将其接种于96孔板后,加入支架混合培养,5d后进行MTT细胞增殖测定。结果:在与含1μg/mL的bFGF支架混合培养,以及与同时含1μg/mL的bFGF、1μg/mL的HGF支架混合培养后,BMSCs增殖明显(P<0.05),但这二组间无统计学差异(P>0.05)。分别与含0.1μg/mL的bFGF支架、0.01μg/mL的bFGF支架、1μg/mL的HGF支架混合培养后,细胞增殖无统计学意义(P>0.05)。结论:壳聚糖-明胶可作为生长因子缓释支架材料;bFGF具有促进BMSCs增殖的作用,促进作用的大小与加入bFGF的量有关;HGF对BMSCs不具有增殖作用;在实验浓度范围内bFGF和HGF体外促进BMSCs增殖上不具有协同性。     

Deconstructed Microfluidic Bone Marrow On‐A‐Chip to Study Normal and Malignant Hemopoietic Cell–Niche Interactions

Human hematopoietic niches are complex specialized microenvironments that maintain and regulate hematopoietic stem and progenitor cells (HSPC). Thus far, most of the studies performed investigating alterations of HSPC‐niche dynamic interactions are conducted in animal models. Herein, organ microengineering with microfluidics is combined to develop a human bone marrow (BM)‐on‐a‐chip with an integrated recirculating perfusion system that consolidates a variety of important parameters such as 3D architecture, cell–cell/cell–matrix interactions, and circulation, allowing a better mimicry of in vivo conditions. The complex BM environment is deconvoluted to 4 major distinct, but integrated, tissue‐engineered 3D niche constructs housed within a single, closed, recirculating microfluidic device system, and equipped with cell tracking technology. It is shown that this technology successfully enables the identification and quantification of preferential interactions—homing and retention—of circulating normal and malignant HSPC with distinct niches.     

Hepatic Differentiation of Human Embryonic Stem Cells as Microscaled Multilayered Colonies Leading to Enhanced Homogeneity and Maturation

Directed differentiation of human embryonic stem cells (hESCs) towards hepatocyte‐like cells on planar tissue culture plates has been extensively investigated with great promise to provide alternative cell sources for drug metabolism/toxicity testing. Recently, hepatic differentiation of hESCs in 3D configuration with better mimicry of embryonic liver development represents incremental efforts to improve the differentiation efficiency and cellular maturation. However, most of the present 3D differentiation configurations involved interruptive operations during the multi‐staged differentiation process, which might impose unwanted influence on cellular differentiation. Most of the current researches resulted in generation of hepatocytes with high expression of AFP, which is minimally expressed in primary hepatocytes. Here, off‐the‐shelf micro‐stencil arrays are developed to generate adherent multilayered colonies composed of hESCs‐derived cells. Uninterrupted cellular differentiation and proliferation is achieved to recapitulate the continuous and multi‐stage liver development. Compared with conventional 2D format, the micro‐scaled multilayered colonies with uniform and defined sizes constrained within the microwells are composed of more homogenous and mature hepatocyte‐like cells with significantly lowered AFP expression and elevated hepatic functions. The multilayered colonies as novel 3D configuration for hepatic differentiation of hESCs represent a significant step toward efficient generation of functional hepatocytes for regenerative medicine and drug discovery.     

Dynamical modelling of haematopoiesis: an integrated view over the system in homeostasis and under perturbation

A very high number of different types of blood cells must be generated daily through a process called haematopoiesis in order to meet the physiological requirements of the organism. All blood cells originate from a population of relatively few haematopoietic stem cells residing in the bone marrow, which give rise to specific progenitors through different lineages. Steady-state dynamics are governed by cell division and commitment rates as well as by population sizes, while feedback components guarantee the restoration of steady-state conditions. In this study, all parameters governing these processes were estimated in a computational model to describe the haematopoietic hierarchy in adult mice. The model consisted of ordinary differential equations and included negative feedback regulation. A combination of literature data, a novel divide et impera approach for steady-state calculations and stochastic optimization allowed one to reduce possible configurations of the system. The model was able to recapitulate the fundamental steady-state features of haematopoiesis and simulate the re-establishment of steady-state conditions after haemorrhage and bone marrow transplantation. This computational approach to the haematopoietic system is novel and provides insight into the dynamics and the nature of possible solutions, with potential applications in both fundamental and clinical research.     

TiO2 Nanorod Array Constructed Nanotopography for Regulation of Mesenchymal Stem Cells Fate and the Realization of Location‐Committed Stem Cell Differentiation

As a physical cue for controlling the fate of stem cells, surface nanotopography has attracted much attention to improve the integration between implants and local host tissues and cells. A biocompatible surface TiO₂ nanorod array is proposed to regulate the fate of bone marrow derived mesenchymal stem cells (MSCs). TiO₂ substrates with different surface nanotopographies: a TiO₂ nanorod array and a polished TiO₂ ceramic are built by hydrothermal and sintering processes, respectively. The assessment of morphology, viability, gene expression, and protein characterization of the MSCs cultured on the different TiO₂ substrates proves that a TiO₂ nanorod array promotes the osteogenic differentiation of MSCs, while a TiO₂ ceramic with a smooth surface suppresses it. Periodically assembled TiO₂ nanorod array stripes on the smooth TiO₂ ceramic are constructed by a combination of microfabrication and a chemical synthesis process, which realizes the location‐committed osteogenic differentiation of MSCs. A route to control the differentiation of MSCs by a nanostructured surface, which can also control the location and direction of MSCs on the surface of biomaterials with micro‐nano scale surface engineering, is demonstrated.