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1.
Niches regulate lineage-specific stem cell self-renewal versus differentiation in vivo and are composed of supportive cells and extracellular matrix components arranged in a three-dimensional topography of controlled stiffness in the presence of oxygen and growth factor gradients. Mimicking stem cell niches in a defined manner will facilitate production of the large numbers of stem cells needed to realize the promise of regenerative medicine and gene therapy. Progress has been made in mimicking components of the niche. Immobilizing cell-associated Notch ligands increased the self-renewal of hematopoietic (blood) stem cells. Culture on a fibrous scaffold that mimics basement membrane texture increased the expansion of hematopoietic and embryonic stem cells. Finally, researchers have created intricate patterns of cell-binding domains and complex oxygen gradients.  相似文献   

2.
Multipotent stem cells in the body facilitate tissue regeneration, growth, and wound healing throughout life. The microenvironment in which they reside provides signals that direct these progenitors to proliferate, differentiate, or remain dormant; these factors include soluble molecules, the extracellular matrix, neighboring cells, and physical stimuli. Recent advances in the culture of embryonic stem cells and adult progenitors necessitate an increased understanding of these phenomena. Here, we summarize the interactions between stem cells and their local environment, drawing on in vivo observations and tissue culture studies. In addition, we describe novel methods of characterizing the effects of various environmental factors and review new techniques that enable scientists and engineers to more effectively direct stem cell fate.  相似文献   

3.
Watts KL  Adair J  Kiem HP 《Cytotherapy》2011,13(10):1164-1171
Hematopoietic stem cell (HSC) gene therapy remains a highly attractive treatment option for many disorders, including hematologic conditions, immunodeficiencies including human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), and other genetic disorders such as lysosomal storage diseases. In this review, we discuss the successes, side-effects and limitations of current gene therapy protocols. In addition, we describe the opportunities presented by implementing ex vivo expansion of gene-modified HSC, as well as summarize the most promising ex vivo expansion techniques currently available. We conclude by discussing how some of the current limitations of HSC gene therapy could be overcome by combining novel HSC expansion strategies with gene therapy.  相似文献   

4.
Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T‐flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex‐1®, by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50 × 106 cell g?1 Cytodex‐1®). Then, the MSC aggregates were put into contact with additional available surfaces (T‐flask, fresh and used Cytodex‐1®) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T‐flasks or fresh Cytodex‐1® carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex‐1® carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex‐1® microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012  相似文献   

5.
Widespread use of embryonic and adult stem cells for therapeutic applications will require reproducible production of large numbers of well-characterized cells under well-controlled conditions in bioreactors. During the past two years, substantial progress has been made towards this goal. Human mesenchymal stem cells expanded in perfused scaffolds retained multi-lineage potential. Mouse neural stem cells were expanded as aggregates in serum-free medium for 44 days in stirred bioreactors. Mouse embryonic stem cells expanded as aggregates and on microcarriers in stirred vessels retained expression of stem cell markers and could form embryoid bodies. Embryoid body formation from dissociated mouse embryonic stem cells, followed by embryoid body expansion and directed differentiation, was scaled up to gas-sparged, 2-l instrumented bioreactors with pH and oxygen control.  相似文献   

6.
Hematopoietic stem cells (HSCs) effectively and continuously replenish the full range of blood-cell populations. Bone-marrow and umbilical-cord blood stem-cell transplantation (SCT) restore hematopoiesis when used in various hematological and oncohematological disorders in adults and children. However, wider clinical application of effective SCT-based approaches is limited by the low number of primitive HSCs in the available biospecimens. Development of effective protocols of HSC expansion in vitro is therefore necessary. In this review, the notion of bone marrow hematopoiesis is discussed as a complex cellular system and a comparative analysis of various methods for HSC expansion in vitro is provided. The review is illustrated by our own data supporting application of various feeder-cell types for human HSC expansion in vitro.  相似文献   

7.
8.
This review provides insight into two clinical trials conducted with ex vivo manipulated CD34+ cells. The first was an attempt to deliver a gene therapy for treatment of HIV and the second an attempt to improve rates of hemopoietic recovery with ex vivo generated myeloid cells.  相似文献   

9.
In the biopharmaceutical industry, adherent growing stem cell cultures gain worldwide importance as cell products. The cultivation process of these cells, such as in stirred tank reactors or in fixed bed reactors, is highly sophisticated. Cultivations need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time on-line monitoring tools are recommended. A tool meeting the new requirements may be the dielectric spectroscopy for online viable cell mass determination by measurement of the permittivity. To establish these tools, proper offline methods for data correlation are required. The cell number determination of adherent cells on microcarrier is difficult, as it requires cell detachment from the carrier, which highly increases the statistical error. As an offline method, a fluorescence assay based on SYBR®GreenI was developed allowing fast and easy total cell concentration determination without the need to detach the cells from the carrier. The assay is suitable for glass carriers used in stirred tank reactor systems or in fixed bed systems, may be suitable for different cell lines and can be applied to high sample numbers easily. The linear dependency of permittivity to cell concentration of suspended stem cells with the dielectric spectroscopy is shown for even very small cell concentrations. With this offline-method, a correlation of the cell concentration grown on carrier to the permittivity data measured by the dielectric spectroscopy was done successfully.  相似文献   

10.
The number and self‐renewal capacity of hematopoietic stem cells (HSCs) are tightly regulated at different developmental stages. Many pathways have been implicated in regulating HSC development in cell autonomous manners; however, it remains unclear how HSCs sense and integrate developmental cues. In this study, we identified an extrinsic mechanism by which HSC number and functions are regulated during mouse puberty. We found that the HSC number in postnatal bone marrow reached homeostasis at 4 weeks after birth. Luteinizing hormone, but not downstream sex hormones, was involved in regulating HSC homeostasis during this period. Expression of luteinizing hormone receptor (Lhcgr) is highly restricted in HSCs and multipotent progenitor cells in the hematopoietic hierarchy. When Lhcgr was deleted, HSCs continued to expand even after 4 weeks after birth, leading to abnormally elevated hematopoiesis and leukocytosis. In a murine acute myeloid leukemia model, leukemia development was significantly accelerated upon Lhcgr deletion. Together, our work reveals an extrinsic counting mechanism that restricts HSC expansion during development and is physiologically important for maintaining normal hematopoiesis and inhibiting leukemogenesis.  相似文献   

11.

Background  

Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons.  相似文献   

12.
Muscle stem cells (MuSCs, satellite cells) are the major contributor to muscle regeneration. Like most adult stem cells, long-term expansion of MuSCs in vitro is difficult. The in vivo muscle regeneration abilities of MuSCs are quickly lost after culturing in vitro, which prevents the potential applications of MuSCs in cell-based therapies. Here, we establish a system to serially expand MuSCs in vitro for over 20 passages by mimicking the endogenous microenvironment. We identified that the combination of four pro-inflammatory cytokines, IL-1α, IL-13, TNF-α, and IFN-γ, secreted by T cells was able to stimulate MuSC proliferation in vivo upon injury and promote serial expansion of MuSCs in vitro. The expanded MuSCs can replenish the endogenous stem cell pool and are capable of repairing multiple rounds of muscle injuries in vivo after a single transplantation. The establishment of the in vitro system provides us a powerful method to expand functional MuSCs to repair muscle injuries.  相似文献   

13.
14.
The aim of this study was to develop a robust, quality controlled and reproducible large-scale culture system using serum-free (SF) medium to obtain vast numbers of embryonic stem (ES) cells as a starting source for potential applications in tissue regeneration, as well as for drug screening studies. Mouse ES (mES) cells were firstly cultured on microcarriers in spinner flasks to investigate the effect of different parameters such as the agitation rate and the feeding regimen. Cells were successfully expanded at agitation rates up to 60 rpm using the SF medium and no significant differences in terms of growth kinetics or metabolic profiles were found between the two feeding regimens evaluated: 50% medium renewal every 24 h or 25% every 12 h. Overall, cells reached maximum concentrations of (4.2 ± 0.4) and (5.6 ± 0.8) ×10(6) cells/mL at Day 8 for cells fed once or twice per day; which corresponds to an increase in total cell number of 85 ± 7 and 108 ± 16, respectively. To have a more precise control over culture conditions and to yield a higher number of cells, the scale-up of the spinner flask culture system was successfully accomplished by using a fully controlled stirred tank bioreactor. In this case, the concentration of mES cells cultured on microcarriers increased 85 ± 15-fold over 11 days. Importantly, mES cells expanded under stirred conditions, in both spinner flask and fully controlled stirred tank bioreactor, using SF medium, retained the expression of pluripotency markers such as Oct-4, Nanog, and SSEA-1 and their differentiation potential into cells of the three embryonic germ layers.  相似文献   

15.
Efforts to develop culture technologies capable of eliciting robust human blood stem cell growth have met with limited success. Considering that adult stem cell cultures are complex systems, comprising multiple cell types with dynamically changing intracellular signalling environments and cellular compositions, this is not surprising. Typically treated as single-input single-output systems, adult stem cell cultures are better described as complex, non-linear, multiple-input multiple-output systems wherein the proliferation of subpopulations of cells leads to the formation of intercellular endogenously secreted protein interaction networks. Genomic and proteomic tools need to be applied to generate high-throughput (and ideally high-content) biological measurements of stem cell culture evolution. Datasets describing cellular interaction networks need to be integrated into predictive models of in vitro stem cell development. Ultimately, such models will serve as a starting point for the rational design of blood stem cell expansion bioprocesses utilizing dynamic system perturbations to achieve the preferential expansion of target cell populations.  相似文献   

16.
17.
Autologous stem cell transplantation (ASCT) is the gold standard therapy for suitable multiple myeloma (MM) patients after induction with high dose therapy. To date, the evidence of a reliable marker of prognosis in these cases remains scarce. Our aim was to evaluate appearance of unrelated atypical serum immunofixation patterns (ASIPs) as a marker of prognosis in MM patients submitted to ASCT. We retrospectively analysed data from 65 patients. Interestingly, we observed that presence of ASIPs was associated with longer progression-free survival and longer overall survival. Our results suggested that presence of ASIPs could be a novel marker of good prognosis in MM patients submitted to ASCT.  相似文献   

18.
Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, offer a potential cell source for research, drug screening, and regenerative medicine applications due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Cell fate decisions are based on components of the surrounding microenvironment, including soluble factors, substrate or extracellular matrix, cell–cell interactions, mechanical forces, and 2D or 3D architecture. Depending on their spatio-temporal context, these components can signal hPSCs to either self-renew or differentiate to cell types of the ectoderm, mesoderm, or endoderm. Researchers working at the interface of engineering and biology have identified various factors which can affect hPSC fate, often based on lessons from embryonic development, and they have utilized this information to design in vitro niches which can reproducibly direct hPSC fate. This review highlights culture systems that have been engineered to promote self-renewal or differentiation of hPSCs, with a focus on studies that have elucidated the contributions of specific microenvironmental cues in the context of those culture systems. We propose the use of microsystem technologies for high-throughput screening of spatial–temporal presentation of cues, as this has been demonstrated to be a powerful approach for differentiating hPSCs to desired cell types.  相似文献   

19.
Embryonic stem (ES) cells have the ability to differentiate in vitro into a wide variety of cell types with potential applications for tissue regeneration. However, a large number of cells are required, thus strengthening the need to develop large-scale systems using chemically defined media for ES cell production and/or controlled differentiation. In the present studies, a stirred culture system (i.e. spinner flask) was used to scale-up mouse ES (mES) cell expansion in serum-containing (DMEM/FBS) or serum-free medium, both supplemented with leukemia inhibitory factor (LIF), using either Cytodex 3 or Cultispher S microcarriers. After 8 days, maximal cell densities achieved were (1.9+/-0.1), (2.6+/-0.7) and 3.5x10(6)cells/mL for Cytodex 3 in DMEM/FBS, Cultispher S in DMEM/FBS and Cultispher S in serum-free cultures, respectively, with fold increases of 38+/-2, 50+/-15 and 70. Both microcarriers were suitable to sustain mES cell expansion, though the macroporous Cultispher S seemed to be advantageous in providing a more protective environment against shear stress forces, which harmful effects are exacerbated in serum-free conditions. Importantly, mES cells expanded under stirred conditions using serum-free medium retained their pluripotency and the ability to commit to the neural lineage.  相似文献   

20.
Lung carcinoma is often incurable and remains the leading cancer killer in both men and women. Recent evidence indicates that tumors contain a small population of cancer stem cells that are responsible for tumor maintenance and spreading. The identification of the tumorigenic population that sustains lung cancer may contribute significantly to the development of effective therapies. Here, we found that the tumorigenic cells in small cell and non-small cell lung cancer are a rare population of undifferentiated cells expressing CD133, an antigen present in the cell membrane of normal and cancer-primitive cells of the hematopoietic, neural, endothelial and epithelial lineages. Lung cancer CD133(+) cells were able to grow indefinitely as tumor spheres in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The injection of 10(4) lung cancer CD133(+) cells in immunocompromised mice readily generated tumor xenografts phenotypically identical to the original tumor. Upon differentiation, lung cancer CD133(+) cells acquired the specific lineage markers, while loosing the tumorigenic potential together with CD133 expression. Thus, lung cancer contains a rare population of CD133(+) cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may provide valuable information to be exploited in the clinical setting.  相似文献   

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