scholarly journals Phenotypic analysis of c-Kit expression in epithelial monolayers derived from postnatal rat pancreatic islets

2004 ◽  
Vol 182 (1) ◽  
pp. 113-122 ◽  
Author(s):  
R Wang ◽  
J Li ◽  
N Yashpal
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Matrix Protein ◽  
Ex Vivo ◽  
Stem Cell Marker ◽  
Haematopoietic Stem Cell ◽  
Extracellular Matrix Protein ◽  
Type I ◽  
Cytokeratin 20 ◽  
Epithelial Monolayers

The limitation of available islets for transplantation is a major obstacle for the treatment of diabetes through islet therapy. However, islet monolayers expanded ex vivo may provide a source of progenitor cells and a model to help understand islet development from precursor cell types. The existence of progenitor cells within the islets is highly likely, yet, to date, no fully defined or characterized postnatal stem cell has been isolated, expanded or marked. Our study evaluates the expression of progenitor markers, including the haematopoietic stem cell marker c-Kit, in epithelial monolayers derived from postnatal rat islets through immunofluorescence and RT-PCR, and the ability of precursor-rich monolayers to reform islet-like structures. Islets formed confluent monolayers when cultured on a type I collagen gel which lacked endocrine phenotypes but were positive for cytokeratin 20 and contained an increased proportion of proliferating c-Kit-expressing cells, with the proportion reaching a maximum of 45+/-6% at 8 weeks of culture. Evaluation of transcription factors at the mRNA level revealed constant PDX-1, ngn3 and Pax4 expression, while undifferentiated cell markers, such as Oct4 and alpha-fetoprotein, were also detected frequently after 4 weeks of culture. Changing the extracellular matrix protein to laminin-rich Matrigel, the monolayers re-formed islet-like clusters that secreted insulin in a glucose-responsive fashion. Our data show that islets can be expanded ex vivo to form epithelial monolayers with rich undifferentiating cell populations that are characterized by cells expressing the progenitor markers. These monolayers are capable of extensive proliferation and retain plasticity to form new islet cells, and c-Kit-expressing cells may play an important role in new islet cluster formation.

scholarly journals Nestin expression and clonal analysis of islet-derived epithelial monolayers: insight into nestin-expressing cell heterogeneity and differentiation potential

2005 ◽  
Vol 184 (2) ◽  
pp. 329-339 ◽  
Author(s):  
R Wang ◽  
J Li ◽  
N Yashpal ◽  
N Gao
Keyword(s):  
Transcription Factor ◽  
Glucose Transporter ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Clonal Analysis ◽  
Stem Cell Marker ◽  
Type I ◽  
Cytokeratin 20 ◽  
Differentiation Potential ◽  
Epithelial Monolayers

There has over the last several years been renewed interest in developing a system for generating new islets and a search for a self-renewing population in the pancreas. In particular, the neural stem cell marker nestin has been implicated as an islet precursor marker and its immunoreactivity has been localized in the islets of Langerhans. This study examines islet-derived epithelial monolayers expanded ex vivo to provide a source of nestin-expressing progenitor cells – a model that will help us understand the role of nestin-expressing cells in islet cell development. When cultured on a type I collagen gel, islets formed confluent monolayers which lacked endocrine phenotypes but were positive for cytokeratin 20 and contained an increased proportion of proliferating nestin-expressing cells, reaching a maximum of 54±10%. Co-expression studies demonstrated that the nestin-positive cells are heterogeneous, with some nestin-expressing cells co-localizing with the transcription factor PDX-1 and glucose transporter type 2 or lack of co-expression with vimentin. When clonal populations of nestin-positive cells were expanded and subjected to a differentiation protocol, only a population that expressed the transcription factor PDX-1 at the mRNA level was capable of re-expressing insulin at the mRNA and protein level. In conclusion, these studies demonstrate that expanded nestin-expressing cells in vitro from islet-derived epithelial monolayers are heterogeneous; clonal analysis of nestin-positive cells reveals that a distinct subpopulation of nestin/PDX-1-expressing cells is capable of forming insulin-producing cells.

scholarly journals Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maike Stahlhut ◽  
Teng Cheong Ha ◽  
Ekaterina Takmakova ◽  
Michael A. Morgan ◽  
Adrian Schwarzer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Cell Lines ◽  
Cell Biology ◽  
Ex Vivo ◽  
Haematopoietic Stem Cell ◽  
Transcriptional Dysregulation ◽  
Conditional Gene Expression ◽  
Exponential Expansion ◽  
Drug Dependent

AbstractRegulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

scholarly journals Ex vivo expansion of human hematopoietic stem and progenitor cells

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6083-6090 ◽  
Author(s):  
Ann Dahlberg ◽  
Colleen Delaney ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Notch Signaling ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Growth Factors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

AbstractDespite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field.

Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

2009 ◽  
Vol 423 (1) ◽  
pp. 53-59 ◽  
Author(s):  
Sebastian Kalamajski ◽  
Anders Aspberg ◽  
Karin Lindblom ◽  
Dick Heinegård ◽  
Åke Oldberg
Keyword(s):  
Matrix Protein ◽  
Collagen Type I ◽  
Full Length ◽  
Extracellular Matrix Protein ◽  
Type I ◽  
Similar System ◽  
Collagen Binding ◽  
Osteoblastic Activity ◽  
Collagen Mineralization

The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10–12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10–12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami™) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).

Abstract 2876: Early Retention and Subsequent Engraftment of Transplanted Progenitor Cells is Improved by Delivery within Collagen Matrix

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yan Zhang ◽  
Marc Lamoureux ◽  
Stephanie Thorn ◽  
Vincent Chan ◽  
Joel Price ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Progenitor Cells ◽  
Pet Imaging ◽  
Ex Vivo ◽  
Collagen Matrix ◽  
Cell Labeling ◽  
Type I ◽  
The Matrix ◽  
Early Retention

Background: To investigate the mechanisms involved in the potentiation of cell therapy by delivery matrices, we evaluated the retention and engraftment of transplanted human circulating progenitor cells (CPCs) injected in a collagen matrix by using in vivo positron emission tomography (PET) imaging, ex vivo biodistribution, and immunohistochemistry. Methods: CPCs were labeled with 18 F-FDG and injected with or without a collagen type I-based matrix in the ischemic hindlimb muscle (IM) of rats (2x10 6 cells; n=15/group). Localization of cells was acquired by PET imaging (15 min) at 150 min post-injection. In addition, radionuclide biodistribution, immunofluorescence, and immunohistochemical examination of transplanted CPCs were performed at up to 14 days. Results: Cell labeling efficiency was CPC-concentration dependent (r=0.61, p <0.001), but not 18 F-FDG-dose dependent. Labeled CPCs exhibited excellent short-term stability and viability. Persistence of 18 F-FDG radioactivity in cells was markedly greater than non-specific retention in the matrix. Wholebody (WB) PET images revealed better CPC retention in the IM and less non-specific leakage to other tissues when CPCs were delivered within the matrix (IM/WB retention ratio of 43.9±8.2%), compared to cells injected alone (22.3±10.4%; p =0.040) and to 18 F-FDG injected with or without the matrix (9.7±5.5% and 11.0±5.5%, respectively; p <0.005). Radioactivity biodistribution confirmed that accumulation was increased (by 92.5%; p =0.024) in the IM and reduced (by 1.1 to 23.8%; p <0.05) in non-specific tissues when cells were injected within the matrix, compared to cells injected alone. Anti-human mitochondria staining showed increased cell retention in the IM with use of matrices (3.0±2.1%) versus cells only (1.9±0.8%; p =0.048). At 14 days the number of CD31 + transplanted human cells was greater (1.6±0.1%) when injected within the matrix than injected alone (0.7±0.1%; p =0.004). Conclusions: Collagen-based delivery matrices improve the early retention of transplanted CPCs, which in turn favors subsequent cell engraftment in the ischemic tissue. This mechanism conferred by the matrix has potential implications for the optimization of cell therapy at the early stages after cell delivery.

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