Establishment of Clonal Colony-Forming Assay System for Pancreatic Stem/Progenitor Cells

2002 ◽  
Vol 11 (5) ◽  
pp. 451-453 ◽  
Author(s):  
Atsushi Suzuki ◽  
Kazunobu Oyama ◽  
Katashi Fukao ◽  
Hiromitsu Nakauchi ◽  
Hideki Taniguchi
Keyword(s):  
Progenitor Cells ◽  
Assay System ◽  
Cell Potential ◽  
Differentiated Cells ◽  
Flow Cytometric ◽  
Stem Cell Potential ◽  
Colony Forming Assay ◽  
Flow Cytometric Cell ◽  
Clonal Assay

Pluripotent stem cells found in a number of organs are usually in small cell populations. However, under adaptive stimulation, they enter the stage of growth and differentiation to compensate for the loss of differentiated cells. To analyze stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are strongly required. In this study, we established a colony-forming assay system for pancreatic stem/progenitor cells in vitro. In this culture condition, they received signals for growth and differentiation, and formed clonal colonies including pancreatic endocrine-lineage cells, such as α and β cells. By combining this culture system with flow cytometric cell sorting, pancreatic stem/progenitor cells will be enriched, and their potential can be analyzed precisely in single cell-based experiments.

scholarly journals iPSC-Derived MSCs Versus Originating Jaw Periosteal Cells: Comparison of Resulting Phenotype and Stem Cell Potential

2020 ◽  
Vol 21 (2) ◽  
pp. 587 ◽  
Author(s):  
Felix Umrath ◽  
Marbod Weber ◽  
Siegmar Reinert ◽  
Hans-Peter Wendel ◽  
Meltem Avci-Adali ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Induced Pluripotent Stem Cell ◽  
Mitochondrial Activity ◽  
Cell Potential ◽  
Differentiation Potential ◽  
Periosteal Cells ◽  
Induced Pluripotent ◽  
Stem Cell Potential ◽  
Promising Source

Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from human jaw periosteal cells (JPCs) by transfection of a self-replicating RNA (srRNA) and subsequent differentiation into functional osteogenic progenitor cells. In order to facilitate the prospective transfer into clinical practice, xeno-free reprogramming and differentiation methods were established. In this study, we compared the properties and stem cell potential of the iMSCs produced from JPC-derived iPSCs with the parental primary JPCs they were generated from. Our results demonstrated, on the one hand, a comparable differentiation potential of iMSCs and JPCs. Additionally, iMSCs showed significantly longer telomere lengths compared to JPCs indicating rejuvenation of the cells during reprogramming. On the other hand, proliferation, mitochondrial activity, and senescence-associated beta-galactosidase (SA-β-gal) activity indicated early senescence of iMSCs. These data demonstrate the requirement of further optimization strategies to improve mesenchymal development of JPC-derived iPSCs in order to take advantage of the best features of reprogrammed and rejuvenated cells.

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.

Isolation of Mouse Pancreatic Ductal Progenitor Cells Expressing CD133 and c-Met by Flow Cytometric Cell Sorting

2007 ◽  
Vol 132 (2) ◽  
pp. 720-732 ◽  
Author(s):  
Yuji Oshima ◽  
Atsushi Suzuki ◽  
Kaneaki Kawashimo ◽  
Momotarou Ishikawa ◽  
Nobuhiro Ohkohchi ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Sorting ◽  
Flow Cytometric ◽  
Flow Cytometric Cell

scholarly journals Analysis of in vitro lymphocyte adhesion and transendothelial migration by fluorescent-beads-based flow cytometric cell counting

Cytometry ◽  
1998 ◽  
Vol 32 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Grietje Molema ◽  
Geert Mesander ◽  
Bart Jan Kroesen ◽  
Wijnand Helfrich ◽  
Dirk K.F. Meijer ◽  
...  
Keyword(s):  
Transendothelial Migration ◽  
Cell Counting ◽  
Lymphocyte Adhesion ◽  
Flow Cytometric ◽  
Fluorescent Beads ◽  
Flow Cytometric Cell

Enhanced Myeloid Progenitor Cell Cycling and Apoptosis in Mice Lacking the Chemokine Receptor, CCR2

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1524-1533 ◽  
Author(s):  
Suzanna Reid ◽  
Alec Ritchie ◽  
Landin Boring ◽  
Jennifa Gosling ◽  
Scott Cooper ◽  
...  
Keyword(s):  
Growth Factors ◽  
Progenitor Cells ◽  
Chemokine Receptor ◽  
Flow Cytometric Analysis ◽  
Targeted Disruption ◽  
Littermate Control ◽  
Myeloid Progenitor ◽  
Flow Cytometric ◽  
Myeloid Progenitor Cells

Abstract Chemokines regulate hematopoiesis in part by influencing the proliferative status of myeloid progenitor cells (MPC). Human MCP-1/murine JE, a myelosuppressive chemokine, specifically binds C-C chemokine receptor 2 (CCR2). Transgenic mice containing a targeted disruption in CCR2 that prevents expression of CCR2 mRNA and protein and have MPC that are insensitive to inhibition by MCP-1 and JE in vitro were assessed for potential abnormalities in growth of bone marrow (BM) and spleen MPC. MPC in both unseparated and c-kit+lin− populations of BM from CCR2-deficient (−/−) mice were in a greatly increased proliferation state compared with CCR2 littermate control (+/+) mice, an effect not apparent with progenitors from spleens of CCR2 (−/−) mice. Increased cycling status of CCR2 (−/−) BM MPC did not result in increased numbers of nucleated cells or MPC in BM or spleens of CCR2 (−/−) mice. Possible reasons for this apparent discrepancy were highlighted by flow cytometric analysis of c-kit+lin− BM cells and colony formation by MPC subjected to delayed addition of growth factors. The c-kit+lin− population of BM cells from CCR2 (−/−) mice had a significantly higher percentage of apoptotic cells than those from CCR2 (+/+) BM. However, elevated apoptosis was not associated with decreased numbers of c-kit+lin− cells. The increased percentage of apoptotic c-kit+lin− cells was due to elevated apoptosis within the c-kitdimlin−, but not the c-kitbrightlin−, subpopulations of cells. Consistent with enhanced apoptosis of phenotypically defined cells, MPC from CCR2 (−/−) BM and purified c-kit+lin− cells demonstrated decreased cell survival in vitro upon delayed addition of growth factors. The data suggest that signals received by CCR2 limit proliferation of progenitor cells in the BM, but also enhance survival of these cells.

Inhibition of trophoblast stem cell potential in chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the mouse

Development ◽  
2002 ◽  
Vol 129 (16) ◽  
pp. 3913-3924 ◽  
Author(s):  
Gary D. Uy ◽  
Karen M. Downs ◽  
Richard L. Gardner
Keyword(s):  
Stem Cell ◽  
Growth Patterns ◽  
Mouse Development ◽  
Cell Potential ◽  
Trophoblast Stem ◽  
Trophoblast Stem Cell ◽  
Stem Cell Potential ◽  
Post Implantation ◽  
Embryonic Ectoderm

At the blastocyst stage of pre-implantation mouse development, close contact of polar trophectoderm with the inner cell mass (ICM) promotes proliferation of undifferentiated diploid trophoblast. However, ICM/polar trophectoderm intimacy is not maintained during post-implantation development, raising the question of how growth of undifferentiated trophoblast is controlled during this time. The search for the cellular basis of trophoblast proliferation in post-implantation development was addressed with an in vitro spatial and temporal analysis of fibroblast growth factor 4-dependent trophoblast stem cell potential. Two post-implantation derivatives of the polar trophectoderm – early-streak extra-embryonic ectoderm and late-streak chorionic ectoderm – were microdissected into fractions along their proximodistal axis and thoroughly dissociated for trophoblast stem cell culture. Results indicated that cells with trophoblast stem cell potential were distributed throughout the extra-embryonic/chorionic ectoderm, an observation that is probably attributable to non-coherent growth patterns exhibited by single extra-embryonic ectoderm cells at the onset of gastrulation. Furthermore, the frequency of cells with trophoblast stem cell potential increased steadily in extra-embryonic/chorionic ectoderm until the first somite pairs formed, decreasing thereafter in a manner independent of proximity to the allantois. Coincident with occlusion of the ectoplacental cavity via union between chorionic ectoderm and the ectoplacental cone, a decline in the frequency of mitotic chorionic ectoderm cells in vivo, and of trophoblast stem cell potential in vitro, was observed. These findings suggest that the ectoplacental cavity may participate in maintaining proliferation throughout the developing chorionic ectoderm and, thus, in supporting its stem cell potential. Together with previous observations, we discuss the possibility that fluid-filled cavities may play a general role in the development of tissues that border them.

Multipotential ability of primitive germ cells from neonatal pig testis cultured in vitro

2009 ◽  
Vol 21 (5) ◽  
pp. 696 ◽  
Author(s):  
Sandeep Goel ◽  
Mayako Fujihara ◽  
Kazuo Tsuchiya ◽  
Yuji Takagi ◽  
Naojiro Minami ◽  
...  
Keyword(s):  
Stem Cell ◽  
Transcription Factors ◽  
Germ Cells ◽  
Cultured Cells ◽  
Primordial Germ Cells ◽  
Primary Cultures ◽  
Cell Potential ◽  
Neonatal Pig ◽  
Stem Cell Potential

Gonocytes are progenitor-type germ cells that arise from primordial germ cells and differentiate further into spermatogonia, thereby initiating spermatogenesis. In the present study, freshly isolated gonocytes were found to have either weak or no expression of pluripotency determining transcription factors, such as POU5F1, SOX2 and C-MYC. Interestingly, the expression of these transcription factors, as well as other vital transcription factors, such as NANOG, KLF4 and DAZL, were markedly upregulated in cultured cells. Cells in primary cultures expressed specific germ cell and pluripotency markers, such as lectin Dolichos biflorus agglutinin (DBA), KIT, ZBTB16, stage-specific embryonic antigen (SSEA-1), NANOG and POU5F1. Using a monoclonal antibody to specifically identify porcine germ cells, the stem cell potential of fresh and cultured cells was determined with a testis xenotransplantation assay. Colonised porcine germ cells were detected only in mouse testes that were either transplanted with fresh testicular cells or with cells from primary cultures. Interestingly, testes transplanted with cells from primary cultures showed colonisation of germ cells in the interstitial space, reflecting their tumourigenic nature. The formation of teratomas with tissues originating from the three germinal layers following the subcutaneous injection of cells into nude mice from primary cultures confirmed their multipotency. The results of the present study may provide useful information for the establishment of multipotent germ stem cell lines from neonatal pig testis.

Chromatin-modifying agents promote the ex vivo production of functional human erythroid progenitor cells

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4632-4641 ◽  
Author(s):  
Pratima Chaurasia ◽  
Dmitriy Berenzon ◽  
Ronald Hoffman
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylase Inhibitors ◽  
Ex Vivo ◽  
Gene Expression Pattern ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Differentiated Cells ◽  
Erythroid Progenitor Cells ◽  
H3 Acetylation

Abstract Presently, blood transfusion products (TPs) are composed of terminally differentiated cells with a finite life span. We have developed an ex vivo–generated TP composed of erythroid progenitor cells (EPCs) and precursors cells. Several histone deacetylase inhibitors (HDACIs) were used in vitro to promote the preferential differentiation of cord blood (CB) CD34+ cells to EPCs. A combination of cytokines and valproic acid (VPA): (1) promoted the greatest degree of EPC expansion, (2) led to the generation of EPCs which were capable of differentiating into the various stages of erythroid development, (3) led to epigenetic modifications (increased H3 acetylation) of promoters for erythroid-specific genes, which resulted in the acquisition of a gene expression pattern characteristic of primitive erythroid cells, and (4) promoted the generation of a TP that when infused into NOD/SCID mice produced mature RBCs containing both human adult and fetal globins as well Rh blood group Ag which persisted for 3 weeks and the retention of human EPCs and erythroid precursor cells within the BM of recipient mice. This ex vivo–generated EPC-TP likely represents a paradigm shift in transfusion medicine because of its potential to continue to generate additional RBCs after its infusion.

scholarly journals Differentiation of normal human pre-B cells in vitro.

1990 ◽  
Vol 172 (1) ◽  
pp. 325-334 ◽  
Author(s):  
J G Villablanca ◽  
J M Anderson ◽  
M Moseley ◽  
C L Law ◽  
R L Elstrom ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
Magnetic Bead ◽  
B Cell Differentiation ◽  
Differentiated Cells ◽  
Flow Cytometric ◽  
Normal Human

The differentiation of surface Ig- pre-B cells into surface Ig+ B cells is a critical transition in mammalian B cell ontogeny. Elucidation of the growth factor requirements and differentiative potential of human pre-B cells has been hampered by the absence of a reproducible culture system that supports differentiation. Fluorescence-activated cell sorting and magnetic bead depletion were used to purify fetal bone marrow CD10+/surface mu- cells, which contain 60-70% cytoplasmic mu+ pre-B cells. CD10+/surface mu- cells cultured for 2 d were observed to differentiate into surface mu+ cells. Analysis by Southern blotting provided direct evidence that rearrangement of kappa light chain genes occurs in culture, and flow cytometric analysis revealed the appearance of surface Ig+ B cells expressing mu/kappa or mu/lambda. Unexpectedly, the kappa/lambda ratio in differentiated cells was the inverse of what is normally observed in adult peripheral blood. Differentiation occurs in the absence of exogenous growth factors or cytokines, suggesting that a stimulus-independent differentiative inertia might characterize pre-B cells in vivo. Future use of this model will facilitate our understanding of normal and abnormal human pre-B cell differentiation.

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