Effect of Small Molecule Supplements during In Vitro Culture of Mouse Zygotes and Parthenogenetic Embryos on Hypoblast Formation and Stem Cell Derivation

2012 ◽  
Vol 8 (4) ◽  
pp. 1088-1097
Author(s):  
K. Versieren ◽  
M. Van der Jeught ◽  
T. O’Leary ◽  
G. Duggal ◽  
J. Gerris ◽  
...  
Keyword(s):  
Stem Cell ◽  
In Vitro Culture ◽  
Small Molecule ◽  
Mouse Zygotes ◽  
Stem Cell Derivation ◽  
Parthenogenetic Embryos

223 GENERATION OF INTERSPECIES CHIMERAS BETWEEN PRIMATE INDUCED PLURIPOTENT STEM CELLS AND PORCINE PARTHENOGENETIC EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 243
Author(s):  
M. Nowak-Imialek ◽  
S. Wunderlich ◽  
D. Herrmann ◽  
S. Klein ◽  
U. Baulain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Culture Conditions ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Parthenogenetic Embryos

The availability of human induced pluripotent stem cell (hiPSC) paves the way to generate regenerative tissue or organs from patient’s own stem cells. The production of chimeric piglets carrying organs that are entirely derived from human stem cells, or at least have a high contribution of human cells or tissues, could be used as a new tissue or an organ replacement in the future treatment of the patients. Here, we produced porcine-nonhuman primate chimeric embryos to assess the feasibility of the potential use of human iPSC for production of human stem cell-derived organs in pigs. Because in vitro culture conditions for cynomolgus monkey iPSC and porcine blastocysts are different, we first identified an effective in vitro culture system for porcine blastocysts and monkey iPSC. We compared blastocyst rates (Days 7 and 8) and number of cells of porcine parthenogenetic blastocysts (Day 8) cultured in 8 different monkey iPSC media and in porcine zygote medium 3 (PZM-3). The best developmental rates of porcine blastocysts were achieved in Knockout DMEM+20% serum replacement monkey medium (iPS 20% medium; N = 65, n = 3). The number of blastocysts on Day 8 cultured in iPS 20% medium was significantly higher (91%; P < 0.05) than in the commonly used porcine PZM-3 medium (65%). We found significantly fewer (P < 0.05) degenerate porcine embryos on Day 8 after culture in iPS 20% medium (9%) compared to PZM-3 (35%). The number of nuclei per blastocyst in iPS 20% medium (88 nuclei; N = 30, n = 3) was significantly higher (P < 0.0001) than in the PZM-3 medium (57 nuclei; N = 54, n = 3). Therefore, we decided to use iPS 20% medium for culture of porcine blastocysts injected with monkey iPSC. Thereafter, we injected clusters of 10 to 15 monkey iPSC transgenic with AAVS1-CAG-Venus into porcine parthenogenetic embryos from Days 4 and 6. Interspecies chimeras were cultured in iPS 20% medium for 24 (for Day 6 embryos) or 48 h (for Day 4 embryos) and observed by confocal microscopy to determine the proportion of Venus-expressing monkey iPSC in porcine embryos. Approximately 37% of blastocysts contained Venus-positive cells after injection of Day 6 embryos (N = 133, n = 4). In contrast, injection into porcine embryos from Day 4 resulted in 73% of Venus-positive blastocysts (N = 69, n = 3). Finally, we investigated proliferation and survival of monkey iPSC in interspecies chimeras after blastocyst plating onto murine fibroblasts. Chimeric blastocyst outgrowth resulted in Venus-expressing monkey iPSC proliferating over 1 week in culture. Outgrowths of all chimeric blastocysts established distinct but separate monkey and porcine stem cell colonies. Here, we optimized the culture conditions for an in vitro interspecies chimera assay in which monkey iPSC are able to survive in porcine embryos. Integration of monkey iPSC to host inner cell mass is relevant for the further contribution to the embryo development. Therefore, to verify this, analysis of cell-cell connection between monkey iPSC and porcine blastocysts and experiments using vivo-derived embryos are currently underway.

scholarly journals Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 659-659
Author(s):  
Kevin A. Goncalves ◽  
Megan D. Hoban ◽  
Jennifer L. Proctor ◽  
Hillary L. Adams ◽  
Sharon L. Hyzy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Small Molecule ◽  
Hdac Inhibitors ◽  
Employment Equity ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (&gt;3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation

2019 ◽  
Vol 55 (7) ◽  
pp. 473-481 ◽  
Author(s):  
Ali Cihan Taskin ◽  
Ahmet Kocabay ◽  
Ayyub Ebrahimi ◽  
Sercin Karahuseyinoglu ◽  
Gizem Nur Sahin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Derivation

scholarly journals In vitro aging of porcine oocytes

2011 ◽  
Vol 49 (No. 3) ◽  
pp. 93-98 ◽  
Author(s):  
I. Petrová ◽  
M. Sedmíková ◽  
E. Chmelíková ◽  
D. Švestková ◽  
R. Rajmon
Keyword(s):  
In Vitro Culture ◽  
Polar Body ◽  
Cell Stage ◽  
Subsequent Aging ◽  
Parthenogenetic Activation ◽  
In Vitro Aging ◽  
Polar Bodies ◽  
Porcine Oocyte ◽  
Parthenogenetic Embryos

Porcine oocytes matured in vitro develop in various ways if they are further cultivated. In our studies these oocytes were cultivated for 1 to 5 days (in vitro aging). During the 1st day of aging, most of them remained at the stage of metaphase II (98%). Then many oocytes underwent the spontaneous parthenogenetic activation. The portion of activated oocytes reached its peak after 2 or 3 days of aging in vitro (39 or 45%). The portion of fragmented oocytes peaked at the same time (28%). During subsequent aging in vitro (i.e. day 4 or 5 of aging), the portion of lysed oocytes significantly increased (30 or 37%). The highest portion of spontaneously activated parthenogenetic embryos at a pronuclear stage (35%) was observed during the 2nd day of aging in vitro. These pronuclear embryos had mainly one polar body with two pronuclei (47% of all pronuclear embryos) or two polar bodies with one pronucleus (38% of all pronuclear embryos). During the 3rd and 5th day of in vitro aging, there was a significant increase in the portion of parthenogenetic embryos cleaved to the 2-cell or 3-cell stage. When considering the prolonged in vitro culture of porcine oocyte, only the first day of aging should be taken into account, since beyond this time significant changes, i.e. parthenogenesis, fragmentation or lysis, occurred in oocytes under in vitro conditions. &nbsp;

Niche-Based Screening Reveals Leukemia Stem Cell Specific Therapeutics

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 760-760
Author(s):  
Kimberly A. Hartwell ◽  
Peter G. Miller ◽  
Alison L. Stewart ◽  
Alissa R. Kahn ◽  
David J. Logan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Drug Discovery ◽  
Small Molecules ◽  
Small Molecule ◽  
High Throughput ◽  
Hematopoietic Cells ◽  
Leukemia Stem Cells

Abstract Abstract 760 Recent insights into the molecular and cellular processes that drive leukemia have called attention to the limitations intrinsic to traditional drug discovery approaches. To date, the majority of cell-based functional screens have relied on probing cell lines in vitro in isolation to identify compounds that decrease cellular viability. The development of novel therapeutics with greater efficacy and decreased toxicity will require the identification of small molecules that selectively target leukemia stem cells (LSCs) within the context of their microenvironment, while sparing normal cells. We hypothesized that it would be possible to systematically identify LSC susceptibilities by modeling key elements of bone marrow niche interactions in high throughput format. We tested this hypothesis by creating and optimizing an assay in which primary murine stem cell-enriched leukemia cells are plated on bone marrow stromal cells in 384-well format, and examined by a high content image-based readout of cobblestoning, an in vitro morphological surrogate of cell health and self-renewal. AML cells cultured in this way maintained their ability to reinitiate disease in mice with as few as 100 cells. 14,720 small molecule probes across diverse chemical space were screened at 5uM in our assay. Retest screening was performed in the presence of two different bone marrow stromal types in parallel, OP9s and primary mesenchymal stem cells (MSCs). Greater than 60% of primary screen hits positively retested (dose response with IC50 at or below 5 μM) on both types of stroma. Compounds that inhibited leukemic cobblestoning merely by killing the stroma were identified by CellTiter-Glo viability analysis and excluded. Compounds that killed normal primary hematopoietic stem and progenitor cell inputs, as assessed by a related co-culture screen, were also excluded. Selectivity for leukemia over normal hematopoietic cells was additionally examined in vitro by comingling these cells on stroma within the same wells. Primary human CD34+ AML leukemia and normal CD34+ cord blood cells were also tested, by way of the 5 week cobblestone area forming cell (CAFC) assay. Additionally, preliminary studies of human AML cells pulse-treated with small molecules ex vivo, followed by in vivo transplantation, provided further evidence of potent leukemia kill across genotypes. A biologically complex functional approach to drug discovery, such as the novel method described here, has previously been thought impossible, due to presumed incompatibility with high throughput scale. We show that it is possible, and that it bears fruit in a first pilot screen. By these means, we discover small molecule perturbants that act selectively in the context of the microenvironment to kill LSCs while sparing stroma and normal hematopoietic cells. Some hits act cell autonomously, and some do not, as evidenced by observed leukemia kill when only the stromal support cells are treated prior to the plating of leukemia. Some hits are known, such as parthenolide and celastrol, and some are previously underappreciated, such as HMG-CoA reductase inhibition. Others are entirely new, and would not have been revealed by conventional approaches to therapeutic discovery. We therefore present a powerful new approach, and identify drug candidates with the potential to selectively target leukemia stem cells in clinical patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Comparison of the Donor Age-Dependent and In Vitro Culture-Dependent Mesenchymal Stem Cell Aging in Rat Model

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Katarzyna Siennicka ◽  
Aleksandra Zołocińska ◽  
Tomasz Dębski ◽  
Zygmunt Pojda
Keyword(s):  
Stem Cell ◽  
In Vitro Culture ◽  
Growth Potential ◽  
Cell Aging ◽  
Donor Age ◽  
Aging Effects ◽  
Age Limit ◽  
Clinical Experiments

Clinical experiments suggest that mesenchymal stem cells (MSCs) may be useful for tissue repair therapies or treatment of the autoimmune disorders. There is still lack of consensus concerning the age limit of MSC donors, majority of researchers suggest the autologous MSC therapies of patients not exceeding age limit of 55-60 yrs. The purpose of our study was to compare the selected parameters of MSCs from adipose tissue (adipose stem cell, ASC) collected from young and old rats of ages corresponding to patient’s ages 25 yrs. and 80 yrs., respectively. The differences of parameters of ASCs from young and old animals were compared with the differences between ASCs from short-term (3 passage) and long-term (30 passage) in vitro culture. Cell morphology, surface marker expression, growth potential, metabolic activity, β-galactosidase activity, clonogenic potential, angiogenic potential, and differentiation ability of ASCs from young and aged animals and from in vitro cultures at 3rd and 30th passages were compared and analyzed. It may be concluded that ASCs may be applied for autologous transplantations in aged patients. Comparison of ASC aging dynamics depending on host aging or in vitro culture duration suggests that long-term in vitro culture may affect ASCs more than natural aging process of their host. We suggest that ASCs expanded in vitro prior to their clinical use must be carefully screened for the possible aging effects resulting not only from donor age, but from the duration of their in vitro culture.

Muscle stem cell isolation and in vitro culture for meat production: A methodological review

2020 ◽  
Author(s):  
Kwang‐Hwan Choi ◽  
Ji Won Yoon ◽  
Minsu Kim ◽  
Hyun Jung Lee ◽  
Jinsol Jeong ◽  
...  
Keyword(s):  
Stem Cell ◽  
In Vitro Culture ◽  
Cell Isolation ◽  
Meat Production ◽  
Muscle Stem Cell ◽  
Methodological Review ◽  
Stem Cell Isolation

scholarly journals Characterization of limbal explant sites: Optimization of stem cell outgrowth in in vitro culture

PLoS ONE ◽  
2020 ◽  
Vol 15 (5) ◽  
pp. e0233075
Author(s):  
Pattama Ekpo ◽  
Naharuthai Inthasin ◽  
Sutthicha Matamnan ◽  
Patimaporn Wongprompitak ◽  
Methichit Wattanapanitch ◽  
...  
Keyword(s):  
Stem Cell ◽  
In Vitro Culture ◽  
Cell Outgrowth
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