Isolation and In Vitro Culture of Human Gut Progenitor Cells

2019 ◽  
pp. 49-62
Author(s):  
Jessica Bruce ◽  
Gerard E. Kaiko ◽  
Simon Keely
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Human Gut

scholarly journals In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid

2014 ◽  
Vol 29 (4) ◽  
pp. 457-469 ◽  
Author(s):  
Federica Riva ◽  
Claudia Omes ◽  
Roberto Bassani ◽  
Rossella E Nappi ◽  
Giuliano Mazzini ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Follicular Fluid ◽  
Culture System ◽  
Mesenchymal Progenitor Cells ◽  
In Vitro Culture System ◽  
Ovarian Follicular Fluid

In Vitro Chemosensitivity of Leukaemic Stem and Progenitor Cells to Gemtuzumab Ozogamicin (Mylotarg) in AML.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 650-650
Author(s):  
Mays Jawad ◽  
Ullas Mony ◽  
Nigel H. Russell ◽  
Monica Pallis
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Preliminary Data ◽  
Gemtuzumab Ozogamicin ◽  
Flow Cytometric Assay ◽  
Total Percentage ◽  
Flow Cytometric ◽  
Cell Kill ◽  
Stem And Progenitor Cells

Abstract Preliminary data from 1115 patients entered into the MRC AML 15 trial indicated that the addition of Gemtuzumab Ozogamicin (GO) to induction chemotherapy improved disease free survival (Abstract #13, ASH 2006). We hypothesised that this improved survival may be underpinned by the specific therapeutic targeting of leukaemic stem and progenitor cells (LSPC). The LSPC subset of AML cells contains those cells capable of self-renewal in culture and of recapitulating leukaemia in animal models. Successful chemotherapeutic targeting of this subset is essential for complete eradication of leukaemia. We have devised a flow cytometric assay which allows us to measure the in vitro chemosensitivity of the LSPC (CD34+CD38-CD123+) subset in as few as 100 cells and we have used the assay to screen the effectiveness of GO against LSPC. CD123 expression is a determining cell surface marker for leukaemic versus normal stem cells and we were able to demonstrate a significant difference in CD123 MFI values between CD34+CD38- of leukaemic (n= 16) versus normal CD34+ CD38- cells (n= 5; p=0.03), demonstrating the sensitivity of our flow cytometric assay in detecting this leukaemic subset. Blast cells from 14 AML samples were treated with GO (10ng/ml) for 48 hours in an in vitro culture system that maintains LSPC viability. A significant reduction in the number of LSPC (n=14; median 46% cell kill; p= 0.002) as well as AML bulk cells (n=14; median 16% cell kill; p= 0.005) was achieved. This data demonstrates the chemosensitivity of AML cells to GO, particularly to the LSPC subset (p=0.001). Also, the total percentage of LSPC at the start of the assay was found to be positively correlated with GO chemosensitivity (p<0.0001) at 48 hours in in vitro culture (n=14). We have extended culture time for up to 96 hours and preliminary data suggest a further achievable LSPC kill (median 51% cell kill; n= 8). CD33 expression in bulk and CD34+ CD38- populations was explored in the same AML patients. Although CD33 MFI values were highly variable (n= 16; Median = 34.82 and range= 3.7 – 116.54 in bulk fraction and median = 13.69 and range= 0.47 – 436.73 in CD34+ CD38- fraction), we found a significant correlation in CD33 MFI values between bulk and CD34+ CD38- cells (p< 0.0001). Also, the total percentage of CD34+CD38-CD33+ cells was found to be positively correlated with LSPC GO chemosensitivity (n= 14; p= 0.04) after 48 hours of in vitro culture. The GO chemosensitivity of mononuclear cells from mobilised healthy donors was investigated and these were found to be insensitive to this agent both at the bulk cell level and in the CD34+ CD38- subset (mean % cell kill of 10% and 5%, respectively; n=3) after 48 hour in vitro culture. This data establishes the specific targeting of GO to CD123+ CD34+ CD38- and CD33+CD34+ CD38- LSPC, while sparing normal stem and progenitor cells. In conclusion, with many novel agents and drug combinations available for research, we have developed an assay for screening drug effectiveness against LSPC and have demonstrated that GO targets this subset effectively. Combination drugs with GO now need to be further investigated for the complete eradication of LSPC.

Characterization of Hematopoietic Stem Cell Frequency and Function In Unexplained Anemia of the Elderly

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2047-2047
Author(s):  
Wendy Pang ◽  
Elizabeth Price ◽  
Irving L. Weissman ◽  
Stanley L. Schrier
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
In Vitro Culture ◽  
Progenitor Cells ◽  
The Elderly ◽  
Elderly Subjects ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
And Function

Abstract Abstract 2047 Anemia is both a highly prevalent and clinically important condition that causes significant morbidity and mortality in the elderly population. While anemia in the elderly can be attributed to a number of causes, approximately 30% of elderly subjects with anemia have no overt etiology and fall under the category of unexplained anemia of the elderly (UA). There is increasing evidence to suggest that changes in the frequency and/or function of hematopoietic stem and progenitor cells may contribute to the onset and pathophysiology of age-associated hematological conditions, such as UA. Hematopoietic stem cells (HSC) reside at the top of the hematopoietic hierarchy and can differentiate, via increasingly committed downstream progenitors, into all the mature cells of the hematopoietic system. Human myelo-erythroid development proceeds through a set of oligopotent progenitors: HSC give rise to multipotent progenitors (MPP), which give rise to common myeloid progenitors (CMP), which in turn give rise to granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP). We use flow cytometry and in vitro culture of sorted human HSC (Lin-CD34+CD38-CD90+CD45RA-), MPP (Lin-CD34+CD38-CD90-CD45RA-), CMP (Lin-CD34+CD38+CD123+CD45RA-), GMP (Lin-CD34+CD38+CD123+CD45RA+), and MEP (Lin-CD34+CD38+CD123-CD45RA-) from hematologically normal young (23 samples; age 20–35) and elderly (11 samples; age 65+) and UA (5 samples; age 65+) bone marrow samples in order to characterize the changes in the distribution and function of hematopoietic stem and progenitor populations during the aging process and, in particular, in the development of UA. We found that UA patients contain higher frequencies of HSC compared to both elderly normal (1.5-fold; p<0.03) and young normal samples (2.8-fold; p<10-5). We also found increased frequencies of MPP from UA patients compared to MPP from elderly normal (2.6-fold; p<0.002) and young normal samples (5.8-fold; p<0.04). While we observed similar frequencies of CMP among the three groups, we found a notable trend suggesting decreased frequencies of GMP and corresponding increased frequencies of MEP in UA patients. Functionally, HSC from the three groups exhibit statistically insignificant differences in the efficiency of colony formation under the myeloid differentiation-promoting methylcellulose-based in vitro culture conditions; however, on average, HSC from elderly bone marrow samples, regardless of the presence or absence of anemia, tend to form fewer colonies in methylcellulose. Interestingly, HSC from UA patients produce more granulocyte-monocyte (CFU-GM) colonies and fewer erythroid (CFU-E and BFU-E) colonies, compared to HSC from normal samples (p<0.001). Similarly, CMP from UA patients, compared to normal CMP, yield skewed distributions of myeloid-erythroid colonies when plated in methylcellulose, significantly favoring production of CFU-GM colonies over CFU-E and BFU-E colonies (p<0.003). Additionally, MEP from UA patients form both CFU-E and BFU-E colonies in methylcellulose albeit at a significantly lower efficiency than MEP from normal bone marrow samples (p<0.01). This is the first study to examine the changes in hematopoietic stem and progenitor populations in UA patients. The changes in the distribution of hematopoietic stem and progenitor cells in UA patients indicate that the HSC and MPP populations, and possibly also the MEP population, expand in the context of anemia, potentially in response to homeostatic feedback mechanisms. Nevertheless, these expanded populations are functionally impaired in their ability to differentiate towards the erythroid lineage. Our data suggest that there are intrinsic defects in the HSC population of UA patients that lead to poor erythroid differentiation, which can be readily observed even in the earliest committed myelo-erythroid progenitors. We have generated gene expression profiling data from these purified hematopoietic stem and progenitor populations from UA patients to try to identify biological pathways and markers relevant to disease pathogenesis and potential therapeutic targets. Disclosures: Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Schrier:Celgene: Research Funding.

Fabrication of a Nanofibrous Scaffold for the In Vitro Culture of Cardiac Progenitor Cells for Myocardial Regeneration

2013 ◽  
Vol 63 (5) ◽  
pp. 229-239 ◽  
Author(s):  
Rouhollah Mehdinavaz Aghdam ◽  
Saeed Shakhesi ◽  
Siamak Najarian ◽  
Mona Malek Mohammadi ◽  
Seyed Hossein Ahmadi Tafti ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Nanofibrous Scaffold ◽  
Myocardial Regeneration ◽  
Cardiac Progenitor Cells

scholarly journals Adult endothelial progenitor cells from human peripheral blood maintain monocyte/macrophage function throughout in vitro culture

Cell Research ◽  
2006 ◽  
Vol 16 (6) ◽  
pp. 577-584 ◽  
Author(s):  
Shi Ju Zhang ◽  
Hao Zhang ◽  
Ying Jie Wei ◽  
Wen Jun Su ◽  
Zhong Kai Liao ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Endothelial Progenitor ◽  
Macrophage Function

scholarly journals Transient and Stable Overexpression of Extracellular Superoxide Dismutase is Positively Associated with the Myogenic Function of Human Skeletal Muscle-Derived Stem/Progenitor Cells

Antioxidants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 817
Author(s):  
Magdalena Nowaczyk ◽  
Agnieszka Malcher ◽  
Agnieszka Zimna ◽  
Wojciech Łabędź ◽  
Łukasz Kubaszewski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
In Vitro Culture ◽  
Progenitor Cells ◽  
Cell Population ◽  
Human Skeletal Muscle ◽  
Culture Conditions ◽  
Myogenic Cell ◽  
Lower Percentage ◽  
Infarction Heart

In the present study, the genetic modification of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) was investigated to identify the optimal protocol for myogenic cell preparation for use in post-infarction heart therapy. We used two types of modifications: GFP-transfection (using electroporation) and SOD3 transduction (using a lentiviral vector). SkMDS/PCs were cultured under different in vitro conditions, including standard (21% oxygen) and hypoxic (3% oxygen), the latter of which corresponded to the prevailing conditions in the post-infarction heart. Transfection/transduction efficacy, skeletal myogenic cell marker expression (CD56), cellular senescence, and apoptosis, as well as the expression of antioxidant (SOD1, SOD2, and SOD3), anti-aging (SIRT1 and FOXO), anti-apoptotic (BCL2), and myogenic (MyoD and MyoG) genes, were evaluated. The percentage of GFP-positive SkMDS/PCs was determined as an indicator of the efficacy of transfection, which reached 55%, while transduction showed better efficiency, reaching approximately 85% as estimated by fluorescence microscopy. The CD56-positive SkMDS/PCs were present in approximately 77% of the tested cells after transient transfection and approximately 96% after transduction. Under standard in vitro culture conditions, the ability of the differentiated, transfected SkMDS/PCs to form myotubes was greater than that of the wild type (WT) cell population (p < 0.001), while the cells transduced with the SOD3 gene exhibited an increase in cell fusion under both standard (p < 0.05) and hypoxic conditions (p < 0.001). In transduced SkMDS/PCs, we observed a positive influence of SOD3 overexpression on cell ageing and apoptosis. We observed an increase in the percentage of young cells under standard (p < 0.05) and hypoxic (p < 0.001) in vitro culture conditions, with a notable decrease in the percentage of senescent and advanced senescent cells in the SOD3-overexpressing cell population detected compared to that observed for the untransduced muscle-derived cells. A lower percentage of apoptotic cells was observed for transduced SkMDS/PCs than that for WT cells under hypoxic in vitro culture conditions. In transiently transfected SkMDS/PCs, we observed significantly higher gene expression levels of SOD2 (almost 40-fold) (p < 0.001) and FOXO (p < 0.05) (approximately 3-fold) under both normoxic and hypoxic culture conditions and of BCL2 under hypoxia compared to those observed in untreated cells (WT). In addition, myogenic genes showed a significant increase in MyoD (almost 18-fold) expression under standard culture conditions (p < 0.0001) and decreased MyoG expression (approximately 2-fold) after transfection (p < 0.05) compared with that detected in the WT skeletal muscle-derived cell control. Taken together, these results demonstrate that SOD3-tranduced skeletal muscle-derived cells may have potential for use in the regenerative treatment of the post-infarction heart.

scholarly journals Embryonic Origin of the Mouse Macrophage

Blood ◽  
1972 ◽  
Vol 39 (6) ◽  
pp. 842-849 ◽  
Author(s):  
M. J. Cline ◽  
M. A. S. Moore
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Peroxidase Activity ◽  
Fetal Liver ◽  
Yolk Sac ◽  
Mouse Macrophage ◽  
Surface Receptors ◽  
Embryonic Origin ◽  
Macrophage Precursors

Abstract Progenitor cells capable of giving rise to functional macrophages in in vitro culture were first detectable in the fetal yolk sac of the mouse between day 7 and 8 of gestation. Macrophage progenitors were not detectable outside the yolk sac until day 11. Although the early yolk sac contained macrophage precursors, no cells with the morphologic or functional characteristics of mouse promonocytes or more mature macrophages were observed. Promonocytes and macrophages were first identified in the 10-day yolk sac and 11-day fetal liver. These cells were characterized by surface receptors for IgG immunoglobulin, peroxidase activity (promonocytes), glass adherence, and phagocytosis of a large yeast particle (macrophages). From these observations, we conclude that the early fetal yolk sac is the embryonic site of origin of the macrophage precursor and that this precursor is "proximal" to the promonocyte on the pathway of sequential macrophage maturation.

In vitro culture studies of granulocyte/macrophage and erythroid progenitor cells in lymphoproliferative disorders

1986 ◽  
Vol 52 (1) ◽  
pp. 553-561 ◽  
Author(s):  
Klaus Geissler ◽  
Wolfgang Hinterberger ◽  
Peter Bettelheim ◽  
Erich Neumann ◽  
Eva R. Grümayer ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Lymphoproliferative Disorders ◽  
Erythroid Progenitor ◽  
Culture Studies ◽  
Erythroid Progenitor Cells

scholarly journals Near-optimal Conditions for the In Vitro Culture of Hemopoietic Progenitor Cells in Bone Marrow from the Rat

2009 ◽  
Vol 37 (2) ◽  
pp. 170-174 ◽  
Author(s):  
Gemma Molyneux ◽  
Sian Rizzo ◽  
John Turton ◽  
Parvinder Phul ◽  
Frances Gibson
Keyword(s):  
Bone Marrow ◽  
In Vitro Culture ◽  
Progenitor Cells ◽  
Optimal Conditions ◽  
Hemopoietic Progenitor ◽  
Hemopoietic Progenitor Cells
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