scholarly journals Microstructured hydrogels to guide self-assembly and function of lung alveolospheres

2021 ◽  
Author(s):  
Claudia Loebel ◽  
Aaron I. Weiner ◽  
Jeremy B. Katzen ◽  
Michael P. Morley ◽  
Vikram Bala ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
3D Culture ◽  
Size Composition ◽  
Mouse Tissue ◽  
Alveolar Type ◽  
Primary Mouse ◽  
Defined Culture

AbstractEpithelial cell organoids have increased opportunities to probe questions on tissue development and disease in vitro and for therapeutic cell transplantation. Despite their potential, current protocols to grow these organoids almost exclusively depend on culture within three-dimensional (3D) Matrigel, which limits defined culture conditions, introduces animal components, and results in heterogenous organoids (i.e., shape, size, composition). Here, we describe a method that relies on polymeric hydrogel substrates for the generation and expansion of lung alveolar organoids (alveolospheres). Using synthetic hydrogels with defined chemical and physical properties, human induced pluripotent stem cell (iPSC)-derived alveolar type 2 cells (iAT2s) self-assemble into alveolospheres and propagate in Matrigel-free conditions. By engineering pre-defined microcavities within these hydrogels, the heterogeneity of alveolosphere size and structure was reduced when compared to 3D culture while maintaining alveolar type 2 cell fate of human iAT2 and primary mouse tissue-derived progenitor cells. This hydrogel system is a facile and accessible culture system for the culture of primary and iPSC-derived lung progenitors and the method could be expanded to the culture of other epithelial progenitor and stem cell aggregates.

Deterministic Trigger of Self-Renewal in Expanded HSCs Expressing Hoxb4 + Antisense Pbx1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 800-800
Author(s):  
Sonia Cellot ◽  
Jana Krosl ◽  
Keith Humphries ◽  
Guy Sauvageau
Keyword(s):  
Cell Proliferation ◽  
Stem Cell ◽  
Cell Fate ◽  
Time Course ◽  
Cell Cycle Distribution ◽  
Self Renewal ◽  
Primary Mouse ◽  
The Impact

Abstract We previously reported the generation of pluripotent and ultracompetitive HSCs through modulation of Hoxb4 and Pbx1 levels. These Hoxb4hiPbx1lo HSCs display a tremendous regenerative potential, yet they are still fully responsive to in vivo regulatory signals that control stem cell pool size (20 000 HSCmouse) and differentiation pathways. Further work in our laboratory attempted to circumvent these physiological constraints by expanding Hoxb4hiPbx1lo transduced HSCs in vitro, and hence revealing their intrinsic expansion potential. Independent experiments were performed where primary mouse BM cells were co-infected with retroviruses encoding antisense Pbx1 cDNA plus YFP, and Hoxb4 plus GFP (double gene transfer ranged between 20–50%). Hoxb4hiPbx1lo HSCs measured using the CRU assay expanded by 105-fold during a 12 day in vitro culture. Following serial transplantations, these cells displayed an additional 4–5 log expansion in vivo. Total stem cell content per animal remained within normal limits. Southern blot analyses of proviral integrations showed that the expansion was polyclonal, and analyses of individually expanded clones provided a molecular proof of in vitro self-renewal (SR). This unprecedented level of HSC expansion in such a short time course (105-fold in 12 days) implies an absolute HSC doubling time of approximately 17 hours in our culture, raising the possibility that virtually all dividing HSCs undergo self-renewal. This analysis prompted us to dissect the impact of Hoxb4 on cell proliferation versus cell fate (SR?). When analyzed during the period of maximal HSC expansion, the cell cycle distribution of Sca+ or Sca+Lin− cells were comparable between the cultures initiated with neo control versus Hoxb4 BM cells (CTL vs Hoxb4: G0/G1: 66% vs 83%; S: 15% vs 9%; G2/M: 18% vs 7%). Correspondingly, CFSE tracking studies confirmed the identical, or even lower, number of cellular divisions in Sca+ cells isolated from cultures initiated with Hoxb4 versus neo transduced cells. Annexin V studies precluded protection from apoptosis as the major mechanism to increase HSC numbers since similar results (3–10% positive cells) were observed in the Hoxb4 versus neo-transduced cells. In summary, our studies support the emerging concept that distinct molecular pathways regulate cell proliferation and self-renewal, suggesting that Hoxb4 + antisense Pbx1 predominantly triggers self-renewal over HSC proliferation.

scholarly journals Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

2020 ◽  
Author(s):  
James A. Gregory ◽  
Emily Hoelzli ◽  
Rawan Abdelaal ◽  
Catherine Braine ◽  
Miguel Cuevas ◽  
...  
Keyword(s):  
Stem Cell ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Mouse Tissue ◽  
Cell Type ◽  
Primary Mouse ◽  
Cell Type Specific

AbstractGenetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease, but also challenge our ability to resolve cell-type specific perturbations. Here we report an extension of the RiboTag system, first developed to achieve cell-type restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables efficient depletion of off-target RNA in mixed species primary co-cultures and in hiPSC-derived neural progenitor cells, motor neurons, and GABAergic neurons. Nonetheless, depletion efficiency varies across independent experimental replicates. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

scholarly journals Cell Type-Specific In Vitro Gene Expression Profiling of Stem Cell-Derived Neural Models

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1406
Author(s):  
James A. Gregory ◽  
Emily Hoelzli ◽  
Rawan Abdelaal ◽  
Catherine Braine ◽  
Miguel Cuevas ◽  
...  
Keyword(s):  
Stem Cell ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Mouse Tissue ◽  
Cell Type ◽  
Primary Mouse ◽  
Cell Type Specific

Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human-induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease but also challenge our ability to resolve cell type-specific perturbations. Here, we report an extension of the RiboTag system, first developed to achieve cell type-restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables depletion of up to 87 percent of off-target RNA in mixed species co-cultures. Nonetheless, depletion efficiency varies across independent experimental replicates, particularly for hiPSC-derived motor neurons. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

Use of 3D culture systems to generate human induced pluripotent stem cell-derived [beta]-cells in vitro

2018 ◽  
Author(s):  
Fantuzzi Federica ◽  
Toivonen Sanna ◽  
Schiavo Andrea Alex ◽  
Pachera Nathalie ◽  
Rajaei Bahareh ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Cells ◽  
Pluripotent Stem Cell ◽  
3D Culture ◽  
Induced Pluripotent Stem Cell ◽  
Culture Systems ◽  
Induced Pluripotent

scholarly journals Functional Specificity of the Members of the Sos Family of Ras-GEF Activators: Novel Role of Sos2 in Control of Epidermal Stem Cell Homeostasis

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2152
Author(s):  
Fernando C. Baltanás ◽  
Cynthia Mucientes-Valdivieso ◽  
L. Francisco Lorenzo-Martín ◽  
Natalia Fernández-Parejo ◽  
Rósula García-Navas ◽  
...  
Keyword(s):  
Stem Cell ◽  
Layer Structure ◽  
3D Culture ◽  
Hair Follicles ◽  
Specific Cell ◽  
Epidermal Stem Cell ◽  
Primary Keratinocytes ◽  
Cell Homeostasis ◽  
Primary Mouse

Prior reports showed the critical requirement of Sos1 for epithelial carcinogenesis, but the specific functionalities of the homologous Sos1 and Sos2 GEFs in skin homeostasis and tumorigenesis remain unclear. Here, we characterize specific mechanistic roles played by Sos1 or Sos2 in primary mouse keratinocytes (a prevalent skin cell lineage) under different experimental conditions. Functional analyses of actively growing primary keratinocytes of relevant genotypes—WT, Sos1-KO, Sos2-KO, and Sos1/2-DKO—revealed a prevalent role of Sos1 regarding transcriptional regulation and control of RAS activation and mechanistic overlapping of Sos1 and Sos2 regarding cell proliferation and survival, with dominant contribution of Sos1 to the RAS-ERK axis and Sos2 to the RAS-PI3K/AKT axis. Sos1/2-DKO keratinocytes could not grow under 3D culture conditions, but single Sos1-KO and Sos2-KO keratinocytes were able to form pseudoepidermis structures that showed disorganized layer structure, reduced proliferation, and increased apoptosis in comparison with WT 3D cultures. Remarkably, analysis of the skin of both newborn and adult Sos2-KO mice uncovered a significant reduction of the population of stem cells located in hair follicles. These data confirm that Sos1 and Sos2 play specific, cell-autonomous functions in primary keratinocytes and reveal a novel, essential role of Sos2 in control of epidermal stem cell homeostasis.

Evaluation of Congenital Neutropenic Disorders by in Vitro Bone Marrow Culture

PEDIATRICS ◽  
1977 ◽  
Vol 59 (5) ◽  
pp. 739-748
Author(s):  
Peter M. Falk ◽  
Kenneth Rich ◽  
Stephen Feig ◽  
E. Richard Stiehm ◽  
David W. Golde ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Severe Disease ◽  
Stem Cell Differentiation ◽  
Peripheral Blood Cell ◽  
Clinical Expression ◽  
Essentially Normal ◽  
Cell Counts

The congenital neutropenias are a heterogeneous group of diseases whose etiology and pathogenesis are largely unknown. We studied nine neutropenic patients from seven families. Evaluation included peripheral blood cell and differential cell counts, epinephrine and typhoid vaccine stimulation studies, Rebuck skin windows, and bone marrow aspirations for morphological assessment and for in vitro culture in liquid suspension and in agar plates. Parallel cultures were set up with and without colony-stimulating activity (CSA), and peripheral leukocytes were assayed for cellular production of CSA. Patients were initially classified on the basis of their clinical course: benign, mild, moderately severe, or severe disease. One patient in the moderately severe group had an immunoglobulin disorder. Morphologically normal mature granulocytes were seen in bone marrow aspirates of two patients, and maturational defects of varying degree were seen in the remaining seven. Colony formation in agar was markedly reduced below normal in three of seven, moderately reduced in two of seven, and greater than normal in two patients. Colonies in six of seven patients consisted exclusively of macrophages. Marrow from all but one of the nine patients demonstrated poor neutrophil development in suspension culture, and addition of CSA did not result in augmented granulocytic proliferation or maturation. A scheme of normal neutrophil maturation is proposed, and the nine patients were categorized according to this scheme. Four patterns of congenital neutropenia emerged: type 1 was the most benign form of disease with essentially normal clinical and in vitro parameters, and a defect considered to be due to a small committed stem cell pool, abnormal release, or excessive utilization peripherally; type 2 had mild disease with presumed defective committed stem cell differentiation along the granulocyte line; type 3 included benign to severe clinical expression with an apparent defect at the level of the committed granulocyte precursor more severe than in type 2; type 4 disease had varied clinical expression but evidence for a defect at the level of the pluripotent stem cell.

scholarly journals Regulation and Directing Stem Cell Fate by Tissue Engineering Functional Microenvironments: Scaffold Physical and Chemical Cues

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Fei Xing ◽  
Lang Li ◽  
Changchun Zhou ◽  
Cheng Long ◽  
Lina Wu ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Cell Fate ◽  
Chemical Cues ◽  
Physical Factors ◽  
Stem Cell Fate ◽  
Physical And Chemical

It is well known that stem cells reside within tissue engineering functional microenvironments that physically localize them and direct their stem cell fate. Recent efforts in the development of more complex and engineered scaffold technologies, together with new understanding of stem cell behavior in vitro, have provided a new impetus to study regulation and directing stem cell fate. A variety of tissue engineering technologies have been developed to regulate the fate of stem cells. Traditional methods to change the fate of stem cells are adding growth factors or some signaling pathways. In recent years, many studies have revealed that the geometrical microenvironment played an essential role in regulating the fate of stem cells, and the physical factors of scaffolds including mechanical properties, pore sizes, porosity, surface stiffness, three-dimensional structures, and mechanical stimulation may affect the fate of stem cells. Chemical factors such as cell-adhesive ligands and exogenous growth factors would also regulate the fate of stem cells. Understanding how these physical and chemical cues affect the fate of stem cells is essential for building more complex and controlled scaffolds for directing stem cell fate.

scholarly journals Cell stemness is maintained upon concurrent expression of RB and the mitochondrial ribosomal protein S18-2

2020 ◽  
Vol 117 (27) ◽  
pp. 15673-15683
Author(s):  
Muhammad Mushtaq ◽  
Larysa Kovalevska ◽  
Suhas Darekar ◽  
Alexandra Abramsson ◽  
Henrik Zetterberg ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ribosomal Protein ◽  
Severe Combined Immunodeficiency ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Ring Finger ◽  
Histone H2a ◽  
Mitochondrial Ribosomal Protein ◽  
Primary Mouse

Stemness encompasses the capability of a cell for self-renewal and differentiation. The stem cell maintains a balance between proliferation, quiescence, and regeneration via interactions with the microenvironment. Previously, we showed that ectopic expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to immortalization of primary fibroblasts, accompanied by induction of an embryonic stem cell (ESC) phenotype. Moreover, we demonstrated interaction between S18-2 and the retinoblastoma-associated protein (RB) and hypothesized that the simultaneous expression of RB and S18-2 is essential for maintaining cell stemness. Here, we experimentally investigated the role of S18-2 in cell stemness and differentiation. Concurrent expression of RB and S18-2 resulted in immortalization ofRb1−/−primary mouse embryonic fibroblasts and in aggressive tumor growth in severe combined immunodeficiency mice. These cells, which express both RB and S18-2 at high levels, exhibited the potential to differentiate into various lineages in vitro, including osteogenic, chondrogenic, and adipogenic lineages. Mechanistically, S18-2 formed a multimeric protein complex with prohibitin and the ring finger protein 2 (RNF2). This molecular complex increased the monoubiquitination of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3-ligase activity of RNF2. Furthermore, we found enrichment of KLF4 at theS18-2promoter region and that theS18-2expression is positively correlated withKLF4levels. Importantly, knockdown of S18-2 in zebrafish larvae led to embryonic lethality. Collectively, our findings suggest an important role for S18-2 in cell stemness and differentiation and potentially also in cancerogenesis.

Dynamic Mechanical Properties Control Adult Stem Cell Fate

2012 ◽  
Author(s):  
Murat Guvendiren ◽  
Jason A. Burdick
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Adult Stem Cell ◽  
Dynamic Mechanical Properties ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Proliferation And Differentiation ◽  
Important Field ◽  
Control Adult

Stem cells respond to many microenvironmental cues towards their decisions to spread, migrate, and differentiate and these cues can be incorporated into materials for regenerative medicine.1 In the last decade, matrix stiffness alone has been implicated in regulating cellular functions such as migration, proliferation and differentiation. With this in mind, a variety of natural and synthetic polymer systems were used in vitro to mimic the elasticity of native tissues. Despite helping to develop this important field and gather valuable information, these substrates are primarily static and lack the dynamic nature that is observed during many cellular processes such as development, fibrosis and cancer. Thus, it is of great interest to temporally manipulate matrix elasticity in vitro to better understand and develop strategies to control these biological processes. In this work, we utilize a sequential crosslinking approach (initial gelation via addition reaction, secondary crosslinking through light-mediated radical polymerization) to fabricate hydrogel substrates that stiffen (e.g., ∼3 to 30 kPa) either immediately or at later times and in the presence of cells. We demonstrate the utility of this technique by investigating the short-term (several minutes to hours) and long-term (several days to weeks) stem cell response to dynamic stiffening

Sign in / Sign up

Export Citation Format

Share Document