ErbB2 activation contributes to de-differentiation of astrocytes into radial glial cells following induction of scratch-insulted astrocyte conditioned medium

Radial glial cells play a significant role in the repair of spinal cord injuries as they exert critical role in the neurogenesis and act as a scaffold for neuronal migration. Our previous study showed that mature astrocytes of spinal cord can undergo a de-differentiation process and further transform into pluripotential neural precursors; the occurrence of these complex events arise directly from the induction of diffusible factors released from scratch-insulted astrocytes. However, it is unclear whether astrocytes can also undergo rejuvenation to revert to a radial glial progenitor phenotype after the induction of scratch-insulted astrocytes conditioned medium (ACM). Furthermore, the mechanism of astrocyte de-differentiation to the progenitor cells is still unclear. Here we demonstrate that upon treating mature astrocytes with ACM for 10 days, the astrocytes exhibit progressive morphological and functional conversion to radial glial cells. These changes include the appearance of radial glial progenitor cells, changes in the immunophenotypical profiles, characterized by the co-expression of nestin, paired homeobox protein (Pax6) and RC2 as well as enhanced capability of multipotential differentiation. Concomitantly, ErbB2 protein level was progressively up-regulated. Thereby these results provide a potential mechanism by which ACM could induce mature astrocytes to regain the profile of radial glial progenitors due to activating the ErbB2 signaling pathways.     

细叶黄芪叶肉原生质体发育早期细胞核的变化

采用常规超薄切片,放射性同位素标记以及图像处理等技术,对细叶黄芪叶肉原生质体发育早期细胞核内的核仁结构、RNA合成和DNA合成等活动进行了研究。结果表明,离体培养后4h,核仁体积开始增大,培养3—5天时,其体积增长了4—5倍。同时,颗粒区(G)与纤维区(DFC)的比值明显上升,培养5天时,G/DFC比值增长近8倍。此时,核仁的纤维中心也增多。用放射性同位素标记的研究结果表明,培养后4h,~3H-尿苷开始掺入,培养24h,其掺入值达最高峰,是刚游离原生质体的近10倍。~3H-胸苷的掺入是在培养48h后才开始的,培养96h达到最高峰。另外,在培养24h内的切片样品中看到,部分原生质体细胞核内存在着由一些纤维组分构成的束状结构,即核内包含物(IN)。猜测它们可能与核骨架中纤维组分有关。最后,本文着重对细叶黄芪叶肉原生质体发育早期细胞内发生的各种结构与组分变化及时间进程进行了系统分析,将叶肉原生质体早期发育过程分为三个有序阶段,并对叶肉原生质体脱分化过程中的细胞壁再生和脱分化机制等问题进行了讨论。     

Reversible differentiation of myofibroblasts by MyoD

Myofibroblasts participate in tissue repair processes in diverse mammalian organ systems. The deactivation of myofibroblasts is critical for termination of the reparative response and restoration of tissue structure and function. The current paradigm on normal tissue repair is the apoptotic clearance of terminally differentiated myofibroblasts; while, the accumulation of activated myofibroblasts is associated with progressive human fibrotic disorders. The capacity of myofibroblasts to undergo de-differentiation as a potential mechanism for myofibroblast deactivation has not been examined. In this report, we have uncovered a role for MyoD in the induction of myofibroblast differentiation by transforming growth factor-β1 (TGF-β1). Myofibroblasts demonstrate the capacity for de-differentiation and proliferation by modulation of endogenous levels of MyoD. We propose a model of reciprocal signaling between TGF-β1/ALK5/MyoD and mitogen(s)/ERK-MAPK/CDKs that regulate myofibroblast differentiation and de-differentiation, respectively. Our studies provide the first evidence for MyoD in controlling myofibroblast activation and deactivation. Restricted capacity for de-differentiation of myofibroblasts may underlie the progressive nature of recalcitrant human fibrotic disorders.     

A novel preadipocyte cell line established from mouse adult mature adipocytes

We have established a novel preadipocyte cell line from mouse adult mature adipocytes. The mature adipocytes were isolated from fat tissues by taking only the floating population of mature fat cells. The isolated mature adipocytes were de-differentiated into fibroblast-like cells. The in vitro studies showed that the cells could re-differentiate into mature adipocytes after over 20 passages. The in vivo transplantation study also demonstrated that the cells had the full potential to differentiate into mature adipocytes, which has not been shown for the 3T3-L1 preadipocyte cell line derived from mouse embryo. We have further analyzed the expression profile of key fat regulatory genes such as the peroxisome proliferator-activated receptorgamma or CCAAT/enhancer-binding protein gene families. We conclude that our cell line could be used as a preferred alternative to 3T3-L1, potentially reflecting the characteristics of mature adipocytes more, since the cell line is actually derived from adult mature adipocytes.     

De-differentiation Response of Cultured Astrocytes to Injury Induced by Scratch or Conditioned Culture Medium of Scratch-Insulted Astrocytes

Our previous reports indicated that astrocytes (ASTs) in injured adult rat spinal cord underwent a process of de-differentiation, and may acquire the potential of neural stem cells (NSCs). However, the AST de-differentiation and transitional rejuvenation process following injury is still largely unclear. The aim of the present study was to determine whether injured in vitro ASTs can re-enter the multipotential-like stem cell pool and regain NSC characteristics, and to further understand the mechanism of AST de-differentiation. We used an in vitro scratch-wound model to evoke astrocytic response to mechanical injury. GFAP and nestin double-labeled indirect immunofluorescence were carried out to characterize these scratched cells at various periods. Western-blot analysis was used to determine the changes of GFAP and nestin expression following injury. Furthermore, the rate of proliferation was determined by immunocytochemical detection of BrdU incorporating cells. These scratch-wound ASTs were cultured with stem cells medium to explore their ability to generate neurospheres and examine the self-renewal and multi-potency of such neurospheres. Moreover, scratched AST culture supernatant as conditioned cultured medium (ACM) was used to investigate if some diffusible factors derived from injured ASTs could induce de-differentiation of AST. The results showed: (1) the nestin positivity first appeared in GFAP-positive cells at the edge of the scratch, subsequently, disseminated into un-insulted zone. The expression of nestin in AST was increased with longer culture, while that of GFAP was decreased. Furthermore, these nestin-immunoreactive ASTs could generate neurospheres, which showed self-renewal and could be differentiated into neurons, ASTs and oligodendrocytes. (2) Scratched ASTs culture supernatant can induce astrocytic proliferation and de-differentiation. These results reveal that the in vitro injured ASTs can de-differentiate into nestin-positive stem/precursor cells, the process of de-differentiation may arise from direct injury or some diffusible factors released from injured ASTs.     

Maturational differences in superficial and deep zone articular chondrocytes

To examine whether differences in chondrocytes from skeletally immature versus adult individuals are important in cartilage healing, repair, or tissue engineering, superficial zone chondrocytes (SZC, from within 100 μm of the articular surface) and deep zone chondrocytes (DZC, from 30%–45% of the deepest un-mineralized part of articular cartilage) were harvested from immature (1–4 months) and young adult (18–36 months) steers and compared. Cell size, matrix gene expression and protein levels, integrin levels, and chemotactic ability were measured in cells maintained in micromass culture for up to 7 days. Regardless of age, SZC were smaller, had a lower type II to type I collagen gene expression ratio, and higher gene expression of SZ proteins than their DZC counterparts. Regardless of zone, chondrocytes from immature steers had higher levels of Sox 9 and type II collagen gene expression. Over 7 days in culture, the SZC of immature steers had the highest rate of proliferation. Phenotypically, the SZC of immature and adult steers were more stable than their respective DZC. Cell surface α5 and α2 integrin subunit levels were higher in the SZC of immature than of adult steers, whereas β1 integrin subunit levels were similar. Both immature and adult SZC were capable of chemotaxis in response to fetal bovine serum or basic fibroblast growth factor. Our data indicate that articular chondrocytes vary in the different zones of cartilage and with the age of the donor. These differences may be important for cartilage growth, tissue engineering, and/or repair. This work was supported in part by the National Chapter of the Arthritis Foundation, the Ira DeCamp Fellowship for Musculoskeletal Research of the Hospital for Special Surgery, the Institute for Sports Medicine Research in New York, and the National Institute of Health grant AR045748 and was conducted in a facility constructed with support from the Research Facilities Improvement Program (grant no. C06-RR12538-01) of the National Center for Research Resources, National Institutes of Health.     

Analytic formulas for discrete stochastic models of cell populations with both differentiation and de-differentiation

Cell differentiation often appears to be a stochastic process particularly in the hemopoietic system. One of the earliest stochastic models for the growth of stem cell populations was proposed by Till et al. in 1964. In this model there are just two cell types: stem cells and specialized cells. At each time step there is a fixed probability that a stem cell differentiates into a specialized cell and a fixed probability that it undergoes mitosis to produce two stem cells. Even though this model is conceptually simple the myriad of possible outcomes has made it difficult to analyse. We present original closed-form expressions for the probability functions and a fast algorithm for computing them. Renewed interest in stem cells has raised questions about the effect de-differentiation has on stem cell populations. We have extended the stochastic model to include de-differentiation and show that even a small amount of de-differentiation can have a large effect on stem cell population growth.