Stem cell culture: mimicking the stem cell niche in vitro

2013 ◽  
pp. 33-68
Author(s):  
Tiago G. Fernandes ◽  
Maria Margarida Diogo ◽  
Joaquim M.S. Cabral
Keyword(s):  
Cell Culture ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Stem Cell Culture ◽  
Cell Niche

Evidence for a Metabolic Stem Cell Niche.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4046-4046 ◽  
Author(s):  
Michael Cross ◽  
Rudiger Alt ◽  
Lydia Schnapke-Hille ◽  
Thomas Riemer ◽  
Dietger Niederwieser
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Culture Media ◽  
Self Renewal ◽  
Cell Culture Media ◽  
Hematopoietic Stem ◽  
Cell Niche

Abstract The hematopoietic stem cell niche presents a localised environment supporting the balanced maintenance, self-renewal and occasional expansion of the stem cell pool. These options are widely assumed to be regulated exclusively by signalling from specific combinations of stroma-bound or soluble ligands. However, a consideration of the rare conditions under which absolute numbers of stem cells increase in vivo as well as the selective pressures acting on regenerative systems during evolution has led us to propose a metabolic component to the stem cell niche which serves to limit cumulative damage, to avoid the selection of potentially oncogenic mutations and to tie symmetric division to slow proliferation. This would mean that traditional cell culture media based on “systemic” substrates such as glucose and glutamine may actively prevent the symmetric amplification of high quality stem cells, offering a possible explanation for the limited success in this area to date. To investigate this possibility, we have examined the effects of range of carbon and energy sources on the proliferation and maintenance of stem and progenitor cells. Our strategy is to screen a wide variety of culture conditions using murine FDCPmix cells, which are non-tumorigenic but have an innate tendency to amplify symmetrically in the presence of IL-3, and then to test key observations in human UCB CD133+ cells provided with SCF, TPO and FLT-3L. In both cell systems, we do indeed find an unusually low requirement for the systemic substrates glucose and glutamine normally included as major energy and carbon sources in cell culture media. Reducing glucose reduces the yield of committed cells from CD133+ cultures without affecting the accumulation of CD133+CD34+cKit+ progenitors. When provided with alternative substrates more likely to reflect a “niche” type environment, FDCPmix cells can be maintained for long periods in media containing only the trace levels of glucose or glutamine derived from dialysed serum, and show improved self-renewal under these conditions. We have also found that raising osmolarity reduces glucose dependence and simultaneously favours the maintenance both of self-renewing CFU (FDCPmix culture) and of CAFCweek13 (CD133+ culture). In parallel, the use of NMR and mass spectrometry techniques to profile intracellular metabolites in self-renewing and differentiating FDCPmix cells reveals a shift in the metabolite balance indicating reduced glycolysis in the early cells. Taken together, these results suggest that hematopoietic stem cells do indeed have remarkable metabolic characteristics consistent with adaptation to a metabolically limiting niche environment. It may therefore be necessary to identify niche substrates and to combine these with the relevant signalling environment in vitro in order to effectively increase stem cell numbers for research, stem cell transplantation and tissue engineering applications.

scholarly journals Long-Term Human Hematopoietic Stem Cell Culture in Microdroplets

Micromachines ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 90
Author(s):  
Pilar Carreras ◽  
Itziar González ◽  
Miguel Gallardo ◽  
Alejandra Ortiz-Ruiz ◽  
Maria Luz Morales ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Stem Cell Niche ◽  
Hematopoietic Stem ◽  
Stem Cell Culture ◽  
Cell Niche

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device. This approach demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double-layered bead composed by an inner layer of alginate and an outer layer of Puramatrix. We also demonstrated the maintenance and expansion of Multiple Myeloma cells in this construction. Here, the presented microfluidic technique is applied to construct a 3D biomimetic model to recapitulate the human hematopoietic stem cell niche using double-layered hydrogel beads cultured in 10% FBS culture medium. In this model, the long-term maintenance of the number of cells and expansion of hHSCS encapsulated in the proposed structures was observed. Additionally, a phenotypic characterization of the human hematopoietic stem cells generated in the presented biomimetic model was performed in order to assess their long-term stemness maintenance. Results indicate that the ex vivo cultured human CD34+ cells from bone marrow were viable, maintained, and expanded over a time span of eight weeks. This novel long-term stem cell culture methodology could represent a novel breakthrough to improve Hematopoietic Progenitor cell Transplant (HPT) as well as a novel tool for further study of the biochemical and biophysical factors influencing stem cell behavior. This technology opens a myriad of new applications as a universal stem cell niche model potentially able to expand other types of cells.

scholarly journals Scaffold-based 3D cellular models mimicking the heterogeneity of osteosarcoma stem cell niche

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Giada Bassi ◽  
Silvia Panseri ◽  
Samuele Maria Dozio ◽  
Monica Sandri ◽  
Elisabetta Campodoni ◽  
...  
Keyword(s):  
Cell Culture ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Drug Testing ◽  
Tumour Microenvironment ◽  
Osteosarcoma Cell ◽  
Cellular Models ◽  
Cell Niche

AbstractThe failure of the osteosarcoma conventional therapies leads to the growing need for novel therapeutic strategies. The lack of specificity for the Cancer Stem Cells (CSCs) population has been recently identified as the main limitation in the current therapies. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing and screening as well as in the study of cell and molecular biology, are affected by a poor in vitro-in vivo translation ability. To overcome these limitations, this work provides two tumour engineering approaches as new tools to address osteosarcoma and improve therapy outcomes. In detail, two different hydroxyapatite-based bone-mimicking scaffolds were used to recapitulate aspects of the in vivo tumour microenvironment, focusing on CSCs niche. The biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched-CSCs were deeply analysed in these complex cell culture models. The results highlight the fundamental role of the tumour microenvironment proving the mimicry of osteosarcoma stem cell niche by the use of CSCs together with the biomimetic scaffolds, compared to conventional 2D culture systems. These advanced 3D cell culture in vitro tumour models could improve the predictivity of preclinical studies and strongly enhance the clinical translation.

MESENCHYMAL TNFR1 EXPRESSION PRESERVES THE COLONIC EPITHELIAL STEM CELL NICHE

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S7-S8
Author(s):  
Safina Gadeock ◽  
Cambrian Liu ◽  
Brent Polk
Keyword(s):  
Stem Cell ◽  
Stem Cell Niche ◽  
Mesenchymal Cells ◽  
Epithelial Stem Cells ◽  
Epithelial Stem Cell ◽  
Long Term Treatment ◽  
Lgr5 Expression ◽  
Cell Niche

Abstract Tumor necrosis factor (TNF) is a highly expressed cytokine in inflammatory bowel disease (IBD). Although TNF can induce colonic epithelial dysfunction and apoptosis, recent studies suggest that TNF signalling promotes epithelial wound repair and stem cell function. Here we investigated the role of TNF receptor 1 (TNFR1) in mediating TNF’s effects on colonic epithelial stem cells, integral to mucosal healing in colitis. We demonstrate that Tnfr1-/- mice exhibit loss in Lgr5 expression (-52%, p<0.02; N=6) compared to wildtype (WT) controls. However, the opposite result was found in vitro, wherein murine Tnfr1-/- colonoids demonstrated a significant increase in Lgr5 expression (66%, p<0.007; N=6) compared to WT colonoids. Similarly, human colonoids treated with an anti-TNFR1 antibody also demonstrated an increase in Lgr5 expression, relative to IgG controls. To resolve the contradiction in the in vivo versus in vitro environment, we hypothesized that mesenchymal TNFR1 expression regulates the epithelial stem cell niche. To determine the relationships between these cell types, we co-cultured WT or Tnfr1-/- colonoids with WT or Tnfr1-/- colonic myofibroblasts (CMFs). We found that epithelial Lgr5 expression was significantly higher (by 52%, p<0.05; N=3) when co-cultured with WT compared to TNFR1-/- myofibroblasts. The loss of TNFR1 expression in vivo increases the number of αSMA+ mesenchymal cells by nearly 56% (N=6) but considerably reduces the pericryptal PDGFRα+ cells, suggesting modifications in mesenchymal populations that contribute to the epithelial stem cell niche. Functionally, primary Tnfr1-/--CMFs displayed PI3k (p<0.001; N=3) and MAPK (p<0.01; N=3)-dependent increases in migration, proliferation, and differentiation, but RNA profiling demonstrated by diminished levels of stem cell niche factors, Rspo3 (-80%, p<0.0001; N=6) and Wnt2b (-63%, p<0.008; N=6) compared to WT-CMFs. Supplementation with 50ng recombinant Rspo3 for 5 d to Lgr5-GFP organoids co-cultured with TNFR1-/--CMFs restored Lgr5 expression to wildtype levels. Therefore, TNFR1-mediated TNF signalling in mesenchymal cells promotes their ability to support an epithelial stem cell niche. These results should motivate future studies of the stem cell niche in the context of long-term treatment with anti-TNF therapies.

scholarly journals Closer to Nature: The Role of MSCs in Recreating the Microenvironment of the Hematopoietic Stem Cell Niche in vitro

2022 ◽  
pp. 1-10
Author(s):  
Patrick Wuchter ◽  
Anke Diehlmann ◽  
Harald Klüter
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Model Systems ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Hematopoietic Stem Cell Niche ◽  
Cell Niche

<b><i>Background:</i></b> The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. <b><i>Summary:</i></b> This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. <b><i>Conclusion:</i></b> Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

scholarly journals Modeling the Prostate Stem Cell Niche: An Evaluation of Stem Cell Survival and Expansion In Vitro

2010 ◽  
Vol 19 (4) ◽  
pp. 537-546 ◽  
Author(s):  
Shona H. Lang ◽  
Elizabeth Anderson ◽  
Robert Fordham ◽  
Anne T. Collins
Keyword(s):  
Stem Cell ◽  
Cell Survival ◽  
Stem Cell Niche ◽  
Stem Cell Survival ◽  
Cell Niche

Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4641-4651 ◽  
Author(s):  
Pankaj Gupta ◽  
Theodore R. Oegema ◽  
Joseph J. Brazil ◽  
Arkadiusz Z. Dudek ◽  
Arne Slungaard ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Stem Cell Niche ◽  
Ex Vivo ◽  
Recent Observation ◽  
Hematopoietic Progenitors ◽  
Platelet Factor ◽  
Cell Niche

Abstract Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P &lt; .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

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