Abstract 300: Pathophysiologically Regulated Gene Expression in Human Stem & Progenitor Cells for Cardiogenic Differentiation and Repair

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Zhilin Chen ◽  
Sean R Hall ◽  
Keith R Brunt ◽  
Zhan Liu ◽  
David A Ademidun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Starting Point ◽  
Inflammatory Stimuli ◽  
Regulated Gene Expression ◽  
Cardiogenic Differentiation ◽  
Therapeutic Genes

Human stem and progenitor cells have emerged as potentially useful substrates for cardiovascular repair through neovascularization and myocardial regeneration. However, efficacy is limited by impedance to stem cell retention, homing and differentiation in hostile microenvironments, as occur in infarcted myocardium. The objective of the current study was to regulate gene function for tailored therapy in post infarct myocardium. Here we show that hypoxic and inflammatory stimuli of the infarct microenvironment regulate a proportional response in gene expression in human endothelial progenitor (EPC) and mesenchymal stem cells (MSC). Highly efficient lentiviral vectors incorporating hypoxia (HRE) and nuclear factor kappa B (NFkB) responsive elements are used to drive transgenes for survival, autologous stem cell homing and cardiogenic differentiation. Utilizing an internal cytomegalovirus promoter deleted lentiviral transfer vector, an HRE-NFkB bicistronic promoter-reporter vector was constructed with a modified internal ribosome entry sequence between green fluorescent protein and luciferase or therapeutic genes. Either hypoxia or inflammation resulted in a seven to ten-fold response of transgene expression assessed by luciferase activity in EPC (hypoxia, 7608±954; inflammation 11492±1384, P<0.01 and P<0.001 vs control 1049±139 respectively, N=6), while combined hypoxic-inflammatory stimuli resulted in a sixty-fold increase of transgene expression (hypoxic-inflammation, 62364±6609, P<0.001 vs control 1049±139, N=6). These results were recapitulated in MSC and with a series of therapeutic genes as determined by transcript, protein expression and activity. Our results demonstrate that regulated vectors provide a proportional response to hostile post-infarct myocardium. Translating cardiovascular regenerative medicine using stem cells requires managing stem cell survival, function and differentiation. Utilizing site-specific pathophysiological cues to auto-regulate reparative and regenerative gene expression, this study is a starting point for sophisticated platforms for patient tailored cell-based cardiogenic therapy.

Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1449-1449
Author(s):  
Naoya Uchida ◽  
Aylin Bonifacino ◽  
Allen E Krouse ◽  
Sandra D Price ◽  
Ross M Fasano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Transgene Expression ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
One Year

Abstract Abstract 1449 Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. Disclosures: No relevant conflicts of interest to declare.

Abstract 250: Adult Cardiac Stem Cells regulation by microRNAs

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Alex J Matsuda ◽  
Ana I Pinheiro ◽  
Andreia Amaral ◽  
Margarida Gama-Carvalho ◽  
Luís Rosário ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Progenitor Cells ◽  
Expression Profile ◽  
Vascular Function ◽  
Expression Profiles ◽  
Control Cell ◽  
Proliferation And Differentiation ◽  
Regulated Gene Expression ◽  
And Function

In recent years stem and progenitor cells have been identified in the heart, changing our understanding and therapeutic approach of heart disease. These cells can proliferate and differentiate in vitro into beating cardiomyocytes, endothelial and vascular smooth muscle cells, providing the potential for tissue regeneration and repair. microRNAs (miRs) have been identified as master switches controlling proliferation and differentiation, and in particular as key regulators of stem cells and cardiac development and function. Modulation of miR-regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnological and therapeutic applications. We have characterized the expression profile of a subset of 100 miRs with reported functions in stem cell and tissue differentiation in mouse Sca1+ cardiac progenitor cells (CPCs). CPC miR expression profiles were compared with bone marrow mesenchymal stem cells (MSCs) and mouse embryonic heart (E=9) to obtain insights into the origins and function of this rare cell population. Within the miR panel that was studied, a subset with relative high expression levels in CPCs was identified, which may act as negative regulators of differentiation and proliferation. Although the overall expression profile of Sca1+ CPCs is closer to BM-MSCs, the most highly expressed miRs in CPCs are distinctive and predicted to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation. Our results provide novel insights into the gene expression networks active in CPCs, which will now be explored in functional studies to identify key regulators of proliferation and differentiation.

scholarly journals A selective cytotoxic adenovirus vector for concentration of pluripotent stem cells in human pluripotent stem cell-derived neural progenitor cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takamasa Hirai ◽  
Ken Kono ◽  
Rumi Sawada ◽  
Takuya Kuroda ◽  
Satoshi Yasuda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Neural Progenitor Cells ◽  
Neural Progenitor ◽  
Sensitive Detection ◽  
Quality And Safety

AbstractHighly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.

A microfluidic platform enables comprehensive gene expression profiling of mouse retinal stem cells

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Shana O Kelley ◽  
Mahmoud Labib ◽  
Brenda Coles ◽  
Mahla Poudineh ◽  
Brendan Innes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Visual Impairment ◽  
Gene Expression Profiling ◽  
Replacement Therapy ◽  
Retinal Degeneration ◽  
Expression Profiling ◽  
Cell Replacement Therapy ◽  
Cell Replacement

Loss of photoreceptors due to retinal degeneration is a major cause of untreatable visual impairment and blindness. Cell replacement therapy, using retinal stem cell (RSC)-derived photoreceptors, holds promise for reconstituting...

scholarly journals Very Small Embryonic-Like Stem Cells, Endothelial Progenitor Cells, and Different Monocyte Subsets Are Effectively Mobilized in Acute Lymphoblastic Leukemia Patients after G-CSF Treatment

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Andrzej Eljaszewicz ◽  
Lukasz Bolkun ◽  
Kamil Grubczak ◽  
Malgorzata Rusak ◽  
Tomasz Wasiluk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Lymphoblastic Leukemia ◽  
Monocyte Subsets ◽  
Endothelial Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Background. Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid progenitor cells. ALL chemotherapy is associated with numerous side effects including neutropenia that is routinely prevented by the administration of growth factors such as granulocyte colony-stimulating factor (G-CSF). To date, the effects of G-CSF treatment on the level of mobilization of different stem and progenitor cells in ALL patients subjected to clinically effective chemotherapy have not been fully elucidated. Therefore, in this study we aimed to assess the effect of administration of G-CSF to ALL patients on mobilization of other than hematopoietic stem cell (HSCs) subsets, namely, very small embryonic-like stem cells (VSELs), endothelial progenitor cells (EPCs), and different monocyte subsets. Methods. We used multicolor flow cytometry to quantitate numbers of CD34+ cells, hematopoietic stem cells (HSCs), VSELs, EPCs, and different monocyte subsets in the peripheral blood of ALL patients and normal age-matched blood donors. Results. We showed that ALL patients following chemotherapy, when compared to healthy donors, presented with significantly lower numbers of CD34+ cells, HSCs, VSELs, and CD14+ monocytes, but not EPCs. Moreover, we found that G-CSF administration induced effective mobilization of all the abovementioned progenitor and stem cell subsets with high regenerative and proangiogenic potential. Conclusion. These findings contribute to better understanding the beneficial clinical effect of G-CSF administration in ALL patients following successful chemotherapy.

scholarly journals Contribution of Resident Stem Cells to Liver and Biliary Tree Regeneration in Human Diseases

2018 ◽  
Vol 19 (10) ◽  
pp. 2917 ◽  
Author(s):  
Diletta Overi ◽  
Guido Carpino ◽  
Vincenzo Cardinale ◽  
Antonio Franchitto ◽  
Samira Safarikia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Human Liver ◽  
Progenitor Cell ◽  
Biliary Tree ◽  
Tree Regeneration ◽  
Multipotent Stem Cells ◽  
Resident Stem Cells ◽  
Stem Cell Niches

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression.

Establishment of human OCT4 as a putative HSP90 client protein : a case for HSP90 chaperoning pluripotency

2015 ◽  
Author(s):  
◽  
Jason Neville Sterrenberg
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
Cell Biology ◽  
Therapeutic Potential ◽  
Stem Cell Biology ◽  
Client Protein ◽  
Hsp90 Inhibition ◽  
Regulated Gene Expression ◽  
Hsp90 Client Protein

The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors.

Abstract 860: Phenotypic Characterization Of Murine And Human Cardiac-resident Progenitor Cells Isolated On Basis Of Aldehyde-dehydrogenase Activity

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Albert Spicher ◽  
Andrea Meinhardt ◽  
Marc-Estienne Roehrich ◽  
Giuseppe Vassalli
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Aldehyde Dehydrogenase ◽  
Adult Mouse ◽  
Heart Cells ◽  
Stem Cell Markers ◽  
Aldh Activity ◽  
Differentiation Potential ◽  
Cellular Property

Identification of stem cells based on hematopoietic stem cell (HSC) surface markers, such as stem cell antigen-1 (Sca-1) and the c-kit receptor, has limited specificity. High aldehyde-dehydrogenase (ALDH) activity is a general cellular property of stem cells shared by HSC, neural, and intestinal stem cells. The presence of cells with high ALDH activity in the adult heart has not been investigated. Methods: Cells were isolated from adult mouse hearts, and from atrial appendage samples from humans with ischemic or valvular heart disease. Myocyte-depleted mouse Sca-1+, and lineage (Lin)-negative/c-kit+ human heart cells were purified with immunomagnetic beads. ALDH-high cells were identified using a specific fluorescent substrate, and sorted by FACS. Cell surface marker analysis was performed by flow cytometry. Results: Myocyte-depleted mouse heart cells contained 4.8+/−3.2% ALDH-high/SSC-low and 32.6+/−1.6% Sca-1+ cells. ALDH-high cells were Lin-negative, Sca-1+ CD34+ CD105+ CD106+, contained small CD44+ (27%) and CD45+ (15%) subpopulations, and were essentially negative for c-kit (2%), CD29, CD31, CD133 and Flk-1. After several passages in culture, ~20% of ALDH-high cells remained ALDH-high. Myocyte-depleted human atrial cells contained variable numbers of ALDH-high cells ranging from 0.5% to 11%, and 4% Lin-negative/c-kit+ cells. ALDH-high cells were CD29+ CD105+, contained a small c-kit+ subpopulation (5%), and were negative for CD31, CD45 and CD133. After 5 passages in culture, the majority of ALDH-high cells remained ALDH-high. Conclusions: Adult mouse and human hearts contain significant numbers of cells with high ALDH activity, a general cellular property that stem cells possess in different organs, and express stem cell markers (Sca-1 and CD34 in the mouse). The immunophenotype of cardiac-resident ALDH-high cells differs from that previously described for bone marrow ALDH-high HSC, and suggests that this cell population may be enriched in mesenchymal progenitors. Analysis of lineage differentiation potential of ALDH-high cells is in progress. ALDH activity provides a new, practical approach to purifying cardiac-resident progenitor cells.

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