scholarly journals Umbilical Cord Tissue as a Source of Young Cells for the Derivation of Induced Pluripotent Stem Cells Using Non-Integrating Episomal Vectors and Feeder-Free Conditions

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 49
Author(s):  
Aisha Mohamed ◽  
Theresa Chow ◽  
Jennifer Whiteley ◽  
Amanda Fantin ◽  
Kersti Sorra ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Umbilical Cord ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Growth Media ◽  
Donor Cells ◽  
Episomal Vectors ◽  
Umbilical Cord Tissue ◽  
Induced Pluripotent

The clinical application of induced pluripotent stem cells (iPSC) needs to balance the use of an autologous source that would be a perfect match for the patient against any safety or efficacy issues that might arise with using cells from an older patient or donor. Drs. Takahashi and Yamanaka and the Office of Cellular and Tissue-based Products (PMDA), Japan, have had concerns over the existence of accumulated DNA mutations in the cells of older donors and the possibility of long-term negative effects. To mitigate the risk, they have chosen to partner with the Umbilical Cord (UC) banks in Japan to source allogeneic-matched donor cells. Production of iPSCs from UC blood cells (UCB) has been successful; however, reprogramming blood cells requires cell enrichment with columns or flow cytometry and specialized growth media. These requirements add to the cost of production and increase the manipulation of the cells, which complicates the regulatory approval process. Alternatively, umbilical cord tissue mesenchymal stromal cells (CT-MSCs) have the same advantage as UCB cells of being a source of young donor cells. Crucially, CT-MSCs are easier and less expensive to harvest and grow compared to UCB cells. Here, we demonstrate that CT-MSCs can be easily isolated without expensive enzymatic treatment or columns and reprogramed well using episomal vectors, which allow for the removal of the reprogramming factors after a few passages. Together the data indicates that CT-MSCs are a viable source of donor cells for the production of clinical-grade, patient matched iPSCs.

216 EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS ANALOGOUS TO INNER CELL MASS-DERIVED LIF-DEPENDENT MOUSE EMBRYONIC STEM CELLS ESTABLISHED FROM THE DOMESTIC PIG, SUS SCROFA

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
B. P. Telugu ◽  
T. Ezashi ◽  
A. Alexenko ◽  
S. Lee ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Umbilical Cord ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Induced Pluripotent

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer. Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.

Dna methylation of manufactured red blood cells from human induced pluripotent stem cells

2017 ◽  
Vol 53 ◽  
pp. S111-S112
Author(s):  
Isabel Dorn ◽  
Claudia Bernecker ◽  
Slave Trajanoski ◽  
Holm Zaehres ◽  
Peter Schlenke ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Red Blood Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Induced Pluripotent

Generation of human induced pluripotent stem cells from a Bombay individual: Moving towards “universal-donor” red blood cells

2010 ◽  
Vol 391 (1) ◽  
pp. 329-334 ◽  
Author(s):  
Ali Seifinejad ◽  
Adeleh Taei ◽  
Mehdi Totonchi ◽  
Hamed Vazirinasab ◽  
Seideh Nafiseh Hassani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Red Blood Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Induced Pluripotent ◽  
Universal Donor

Hematopoietic Differentiation of Human Induced Pluripotent Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 731-731
Author(s):  
Kyung-Dal Choi ◽  
Junying Yu ◽  
Kimberly Smuga-Otto ◽  
Jessica Dias ◽  
Giorgia Salvagiotto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Differential Expression ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Hematopoietic Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
Differentiation Potential ◽  
Induced Pluripotent

Abstract Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In the present study, we employed an OP9 differentiation system to characterize the hematopoietic differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC; H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs in coculture with OP9 generated all types of colony forming cells (CFCs) as well as CD34+ cells that can be separated into distinct subsets based on differential expression of CD43 and CD31. CD34+CD31+CD43− cells obtained from all iPSCs expressed molecules present on endothelial cells and readily formed a monolayer when placed in endothelial conditions, while hematopoietic CFC potential was restricted to CD43+ cells. iPSC-derived CD43+ cells could be separated into three major subsets based on differential expression of CD235a/CD41a and CD45: CD235a+CD41a+/− (erythro-megakaryocytic progenitors), and lin-CD34+CD43+CD45− (multipotent), and lin-CD34+CD43+CD45+ (myeloid-skewed) primitive hematopoietic cells. Both subsets of primitive hematopoietic cells expressed genes associated with myeloid and lymphoid development, although myeloid genes were upregulated in CD45+ cells, which are skewed toward myeloid differentiation. Cytogenetic analysis demonstrated that iPSCs and derived from them CD43+ cells maintained normal karyotype. In addition short tandem repeat analysis of CFCs generated from IMR90-1 cells has been performed to confirm that blood cells are in fact derived from reprogrammed IMR90 cells, and not from contaminating hESCs. While we observed some variations in the efficiency of hematopoietic differentiation between different iPSCs, the pattern of differentiation was very similar in all seven tested iPSC and five hESC lines. Using different cytokine combinations and culture conditions we were able to expand iPSC-derived myeloid progenitors and induce their differentiation toward red blood cells, neutrophils, eosinophils, macrophages, ostoeclasts, dendritic and Langerhans cells. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and to identify molecules that can correct affected genetic networks.

scholarly journals Advanced Feeder-Free Generation of Induced Pluripotent Stem Cells Directly From Blood Cells

2014 ◽  
Vol 3 (12) ◽  
pp. 1402-1409 ◽  
Author(s):  
Ras Trokovic ◽  
Jere Weltner ◽  
Ken Nishimura ◽  
Manami Ohtaka ◽  
Mahito Nakanishi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Free Generation ◽  
Induced Pluripotent

scholarly journals PSC-RED, an Albumin-Free Robust Erythroid Differentiation Method to Produce Enucleated Red Blood Cells from Human Pluripotent Stem Cells

2019 ◽  
Author(s):  
Emmanuel N Olivier ◽  
Shouping Zhang ◽  
Zi Yan ◽  
Sandra Suzuka ◽  
Karl Roberts ◽  
...  
Keyword(s):  
Stem Cells ◽  
Red Blood Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Erythroid Differentiation ◽  
Economic Viability ◽  
List Type ◽  
Potential Applications ◽  
Induced Pluripotent

AbstractCultured red blood cells (cRBCs) have many potential applications in transfusion medicine and drug delivery. We report that we have developed chemically defined, albumin-free Robust Erythroid Differentiation (RED) methods to produce enucleated cRBCs from human induced pluripotent stem cells (iPSCs). Human iPSC-derived cRBCs produced with either the short or long variation of the RED protocol respectively express embryonic/fetal or a mixture of fetal and adult hemoglobins. The long version of the protocol produces up to 50% of enucleated cells at an unprecedented yield. RED is scalable and relies on inexpensive components and therefore dramatically increases the feasibility and economic viability of all translational applications of cRBCs.HighlightsPSC-RED: A chemically-defined, albumin-free Robust Erythroid Differentiation (RED) methods to produce cRBCs from human induced pluripotent stem cells.PSC-RED produces up to 50% enucleated cells at an unprecedented yield.PSC-RED is scalable and relies on inexpensive components and therefore increases the feasibility and economic viability of translational applications of cRBCs.

Induced Pluripotent Stem Cells and Erythrocyte Production

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-38-SCI-38
Author(s):  
Igor Slukvin
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Hematopoietic Progenitors ◽  
Adult Hemoglobin ◽  
Equity Ownership ◽  
Induced Pluripotent

Abstract Abstract SCI-38 Induced pluripotent stem cells (iPSCs) are somatic cells that have been turned into embryonic-like stem cells by forced expression of factors critical for establishing pluripotency. Because iPSCs can be differentiated into any type of cell in the human body, including hematopoietic cells, they are seen as a logical alternative source of red blood cells (RBCs) for transfusion. In addition, the unlimited expansion potential of iPSCs makes it easy to adopt iPSC technology for RBC biomanufacturing. iPSCs can be generated from any type of donor, including O/Rh-negative universal donors and donors with very rare blood phenotypes, which makes it possible to generate blood products to accommodate virtually all patient groups. We have developed an approach for generating large quantities of RBCs from iPSCs by inducing them to differentiate into CD34+CD43+ hematopoietic progenitors in coculture with OP9 stromal cells, followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. Erythroid cultures produced by this approach consist of leukocyte-free populations of CD235a+ RBCs with robust expansion potential and long (up to 90 days) life spans. In these cultures, up to 1.8×105 RBCs can be generated from a single iPSC. Similar to embryonic stem cells, iPSC-derived RBCs express predominantly embryonic and fetal hemoglobin, with very little adult hemoglobin. It is already feasible to adopt iPSC technologies for producing cGMP-grade RBCs using defined animal-product-free differentiation conditions. However, the induction of the complete switch from embryonic to fetal and adult hemoglobin, as well as the terminal maturation and enucleation of iPSC-derived erythroid cells, remains a significant challenge. We recently identified at least three distinct waves of hematopoietic progenitors with erythroid potential in iPSC differentiation cultures. The characterization of erythroid cells produced from these waves of hematopoiesis may help to define populations with definitive erythroid potential and facilitate the production of erythrocytes from iPSCs. Additional critical steps toward translating iPSC-based RBC technologies to the clinic include the development of bioreactor-based-technology for further scaling-up of cell production, and evaluation of the therapeutic potential and safety of human pluripotent stem cell-derived blood cells in animal models. Overall, the manufacturing of RBCs provides several advantages. It can improve the continuity of the blood supply, minimize/eliminate the risk of infection transmission, reduce the incidence of hemolytic and nonhemolytic transfusion reactions, and provide an opportunity to generate RBCs that fit specific clinical needs by using genetically engineered iPSCs or iPSCs with rare blood groups. Disclosures: Slukvin: CDI: Consultancy, Equity Ownership; Cynata: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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