scholarly journals In vitro clonal analysis of murine pluripotent stem cells isolated from skeletal muscle and adipose stromal cells

2008 ◽  
Vol 36 (2) ◽  
pp. 224-234 ◽  
Author(s):  
Jamie Case ◽  
Tamara L. Horvath ◽  
Christopher B. Ballas ◽  
Keith L. March ◽  
Edward F. Srour
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Clonal Analysis ◽  
Adipose Stromal Cells

Clonal Multilineage Differentiation of Murine Common Pluripotent Stem Cells Isolated from Skeletal Muscle and Adipose Stromal Cells

2005 ◽  
Vol 1044 (1) ◽  
pp. 183-200 ◽  
Author(s):  
JAMIE CASE ◽  
TAMARA L. HORVATH ◽  
JONATHAN C. HOWELL ◽  
MERVIN C. YODER ◽  
KEITH L. MARCH ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Multilineage Differentiation ◽  
Adipose Stromal Cells

Δημιουργία, in vitro πολλαπλασιασμός και διαφοροποίηση ανθρώπινων επαγόμενων πολυδύναμων βλαστοκυττάρων

2016 ◽  
Author(s):  
Ιωάννα Βαρελά
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Rt Pcr ◽  
Induced Pluripotent

Η ανακάλυψη της μεθόδου του κυτταρικού επαναπρογραμματισμού ανθρώπινων δερματικών ινοβλαστών σε επαγόμενα πολυδύναμα βλαστοκύτταρα (induced pluripotent stem cells, iPSCs) το 2007 άνοιξε το δρόμο για τη μελέτη και την εξατομικευμένη θεραπεία πολλών χρόνιων νόσων. Επιδιώξαμε να δημιουργήσουμε iPS - κυτταρικές σειρές επαναπρογραμματίζοντας μεσεγχυματικά στρωματικά κύτταρα (mesenchymal stromal cells, MSCs) μυελού των οστών, μέσω μιας μεθόδου επαναπρογραμματισμού χωρίς ενσωμάτωση γονιδίων στο γενετικό υλικό των κυττάρων. Δερματικοί ινοβλάστες από φυσιολογικούς δότες και μεσεγχυματικά στρωματικά κύτταρα μυελού των οστών από φυσιολογικό δότη μεταμόσχευσης μυελού των οστών και από ασθενή με β-Μεσογειακή αναιμία (β-ΜΑ) διαμολύνθηκαν, μέσω λιποσωματικών φορέων, με συνθετικά mRNA που κωδικοποιούν τους μεταγραφικούς παράγοντες Oct4, Klf4, Sox2, Lin28, c-Myc. Στη συνέχεια, τα κύτταρα ελέγχθηκαν σε καλλιέργειες για τον σχηματισμό αποικιών πολυδύναμων βλαστοκυττάρων. Οι αποικίες απομονώθηκαν και με συνεχείς ανακαλλιέργειες δημιουργήθηκαν κυτταρικές σειρές, οι οποίες εξετάστηκαν για την πολυδυναμία τους με μεθόδους ανίχνευσης της έκφρασης των μεταγραφικών παραγόντων πολυδυναμίας (κυτταρομετρία ροής, RT-PCR, μελέτη του μεταγραφώματος με RNA μικροσυστοιχίες). Ως θετικός μάρτυρας και μέτρο σύγκρισης χρησιμοποιήθηκε πολύ καλά χαρακτηρισμένη εμβρυονική σειρά πολυδύναμων βλαστοκυττάρων. Οι iPS-κυτταρικές σειρές μελετήθηκαν, επίσης, ως προς τη λειτουργική τους πολυδυναμία με τον έλεγχο της ικανότητας τους να δημιουργούν in vitro εμβρυϊκά σωματίδια και in vivo τερατώματα μετά από υποδόρια εμφύτευση τους σε ανοσοανεπαρκείς ποντικούς, και ως προς τη δυνατότητα διαφοροποίησής τους σε αιμοποιητικά προγονικά κύτταρα. Η γενετική σταθερότητα των κυτταρικών σειρών ελέγχθηκε με DNA μικροσυστοιχίες συγκριτικού γονιδιωματικού υβριδισμού (aCGH). Απομονώθηκαν 3 iPS κυτταρικές σειρές από κάθε δείγμα κυττάρων, οι οποίες εμφανίζουν μεταγράφωμα πανομοιότυπο με εκείνο των πολυδύναμων εμβρυονικών βλαστοκυττάρων και. δημιουργούν εμβρυϊκά σωματίδια in vitro και τερατώματα in vivo, τα οποία αποτελούνται από ιστούς καταγωγής και από τα τρία βλαστικά δέρματα. Τα iPSCs των κυτταρικών σειρών πολλαπλασιάζονται για μεγάλο χρονικό διάστημα χωρίς μορφολογικές ενδείξες διαφοροποίησης. Με τη μέθοδο aCGH, στις iPS κυτταρικές σειρές μετά την 10η ανακαλλιέργεια ανιχνεύθηκαν πολυμορφισμοί στον αριθμό αντιγράφων (CNVs), τα οποία ήταν ελλείμματα μεγέθους περίπου 3 Mb. Η διαφοροποίηση των iPSCs σε αιμοποιητικά προγονικά κύτταρα οδήγησε στην παραγωγή CD34+ κυττάρων σε ποσοστό 8-10% των παραχθέντων κυττάρων με ασθενούς έντασης συνέκφραση του CD45, προσομοιάζοντας στο αιμαγγειακό στελεχιαίο κύτταρο. Στην παρούσα διατριβή παρουσιάζεται, για πρώτη φορά στην Ελλάδα, εξ όσων γνωρίζουμε, η τεχνολογία παραγωγής ανθρώπινων iPSCs με μια ασφαλή και αξιόπιστη μέθοδο. Οι iPSCs-κυτταρικές σειρές μπορεί να χρησιμοποιηθούν στη μελέτη ασθενειών, στον έλεγχο φαρμάκων και στην ανάπτυξη πρωτοκόλλων ιστικής μηχανικής και κυτταρικής θεραπείας.

scholarly journals Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion

2019 ◽  
Vol 20 (16) ◽  
pp. 3932 ◽  
Author(s):  
Barbara Świerczek-Lasek ◽  
Jacek Neska ◽  
Agata Kominek ◽  
Łukasz Tolak ◽  
Tomasz Czajkowski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Muscle Development ◽  
Embryonic Stem ◽  
Interleukin 4 ◽  
Myogenic Cells ◽  
The Impact

Pluripotent stem cells convert into skeletal muscle tissue during teratoma formation or chimeric animal development. Thus, they are characterized by naive myogenic potential. Numerous attempts have been made to develop protocols enabling efficient and safe conversion of pluripotent stem cells into functional myogenic cells in vitro. Despite significant progress in the field, generation of myogenic cells from pluripotent stem cells is still challenging—i.e., currently available methods require genetic modifications, animal-derived reagents, or are long lasting—and, therefore, should be further improved. In the current study, we investigated the influence of interleukin 4, a factor regulating inter alia migration and fusion of myogenic cells and necessary for proper skeletal muscle development and maintenance, on pluripotent stem cells. We assessed the impact of interleukin 4 on proliferation, selected gene expression, and ability to fuse in case of both undifferentiated and differentiating mouse embryonic stem cells. Our results revealed that interleukin 4 slightly improves fusion of pluripotent stem cells with myoblasts leading to the formation of hybrid myotubes. Moreover, it increases the level of early myogenic genes such as Mesogenin1, Pax3, and Pax7 in differentiating embryonic stem cells. Thus, interleukin 4 moderately enhances competence of mouse pluripotent stem cells for myogenic conversion.

Perivascular/Mesenchymal Stem Cells from Multiple Human Tissues Support Hematopoiesis in Vitro.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1361-1361
Author(s):  
Elisa Montelatici ◽  
Gabriella Andriolo ◽  
Mihaela Crisan ◽  
Rosaria Giordano ◽  
Paolo Rebulla ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stromal Cells ◽  
Marrow Stromal Cells ◽  
Hematopoietic Cell ◽  
Human Tissues

Abstract Mesenchymal stem cells (MSC) can be derived selectively in culture from multiple organs, an omnipresence we have recently suggested to be explained by the perivascular location of native MSC ancestors within intact tissues (Crisan et al. 2008, in press). We have now analyzed the ability of MSC extracted pro- or retrospectively from different human tissues to support hematopoiesis. MSC were either classically derived in primary cultures of umbilical cord blood (UCB) lineage-depleted mononuclear cells (n=3) or enzymatically dissociated adult adipose tissue (n=3), or grown as CD146+ NG2+ CD34-CD56- CD45- pericytes (n=2) purified by flow cytometry from fetal skeletal muscle and cultured over the long term. In both settings, identical MSC were obtained that maintained a stable CD146+ CD90+ CD73+ CD105+ CD34- CD45- surface phenotype and could differentiate into skeletal muscle, fat, bone and cartilage. CD34+ hematopoietic progenitors (n=3) immunoselected from term UCB were seeded (5×10e3cells/cm2 in triplicate) onto confluent irradiated layers of MSC derived from UCB, adipose tissue or fetal muscle pericytes (MSCu, MSCa and MSCmp, respectively) or, as a control, MS5 bone marrow stromal cells that allow the proliferation of very primitive human progenitor cells. All studies were approved by the relevant institutional regulatory board. The cells were cocultured for 5 weeks in a classical long-term culture-initiating cell assay in a complete medium (MyeloCult H5100, Stem Cell Technologies) containing hydrocortisone but no added cytokine. Wells were scored daily for the presence of cobblestone areas (CA) and half of the medium was replaced every week. Eventually, trypsinized cells from each well were characterized by flow cytometry for the expression of hematopoietic cell markers and assayed for CFC potential. After 14 days of incubation, colonies grown in semi-solid medium were scored as derived from colony forming units (CFU)-granulocyte, erythroid, macrophage, megakaryocyte (GEMM) and as high-proliferative-potential colony precursors (HPPC), the most primitive hematopoietic cell so far identified in a clonogenic assay in vitro. Within the CD45+ gate, all trypsinized cultures contained comparable percentages of CD34+lin- cells (MSCu: 51±9%; MSCa: 58±14%; MSCmp: 61±19%; MS5: 59±18%), the most immature hematopoietic cell compartment maintained during the long-term coculture. MSCu and MSCmp supported a similar cell proliferation during the whole culture while on MSCa, CA formed very rapidly and consistently but eventually decreased over the long-term culture. Interestingly, MSCu and MSCmp supported the development of the highest numbers of HPPC and of CFU giving rise to the largest GEMM colonies, as compared to MSCa that gave the same results as the control MS5 cell line. In summary, all MSCs tested were able to support hematopoiesis and CA formation, albeit with differences in growth kinetics and morphology of the colonies. Herein we show for the first time that purified human perivascular cells exhibit robust hematopoiesis support in vitro, in addition to multilineage mesodermal developmental potential. In conclusion, we demonstrate that MSC from novel sources distinct from the bone marrow are able to support hematopoiesis. These results further sustain the identity, beyond acronyms, between marrow stromal cells, long known for their support of hematopoiesis, and mesenchymal stem cells that gained more recent credit in the field of regenerative medicine because of their multilineage differentiation potential.

scholarly journals Directed Differentiation of Human Pluripotent Stem Cells toward Skeletal Myogenic Progenitors and Their Purification Using Surface Markers

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2746
Author(s):  
Nasa Xu ◽  
Jianbo Wu ◽  
Jose L. Ortiz-Vitali ◽  
Yong Li ◽  
Radbod Darabi
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Surface Markers ◽  
Gene Correction ◽  
Muscle Disorders

Advancements in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) have provided a strong framework for in vitro disease modeling, gene correction and stem cell-based regenerative medicine. In cases of skeletal muscle disorders, iPSCs can be used for the generation of skeletal muscle progenitors to study disease mechanisms, or implementation for the treatment of muscle disorders. We have recently developed an improved directed differentiation method for the derivation of skeletal myogenic progenitors from hiPSCs. This method allows for a short-term (2 weeks) and efficient skeletal myogenic induction (45–65% of the cells) in human pluripotent stem cells (ESCs/iPSCs) using small molecules to induce mesoderm and subsequently myotomal progenitors, without the need for any gene integration or modification. After initial differentiation, skeletal myogenic progenitors can be purified from unwanted cells using surface markers (CD10+CD24−). These myogenic progenitors have been extensively characterized using in vitro gene expression/differentiation profiling as well as in vivo engraftment studies in dystrophic (mdx) and muscle injury (VML) rodent models and have been proven to be able to engraft and form mature myofibers as well as seeding muscle stem cells. The current protocol describes a detailed, step-by-step guide for this method and outlines important experimental details and troubleshooting points for its application in any human pluripotent stem cells.

In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells from Induced Pluripotent Stem Cells through Hemogenic Endothelium

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3866-3866
Author(s):  
Masao Tsukada ◽  
Satoshi Yamazaki ◽  
Yasunori Ota ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Teratoma Formation

Abstract Introduction Generation of engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has long been thought an ultimate goal in the field of hematology. Numerous in vitro differentiation protocols, including trans-differentiation and forward programming approaches, have been reported but have so far failed to generate fully functional HSCs. We have previously demonstrated proof-of-concept for the in vivo generation of fully functional HSCs from induced PSCs (iPSCs) through teratoma formation (Suzuki et al., 2013). However, this method is time-consuming (taking over two months), HSCs are generated at low frequencies, and additionally require co-injection on OP9 stromal cells and SCF/TPO cytokines. Here, we present optimization of in vivo HSC generation via teratoma formation for faster, higher-efficiency HSC generation and without co-injection of stromal cells or cytokines. Results First, we screened reported in vitro trans-differentiation and forward programming strategies for their ability to generate HSCs in vivo within the teratoma assay. We tested iPSCs transduced with the following dox-inducible TF overexpression vectors: (1) Gfi1b, cFOS and Gata2 (GFG), which induce hemogenic endothelial-like cells from fibroblast (Pereira et al.,2013); (2) Erg, HoxA9 and Rora (EAR), which induce short-term hematopoietic stem/progenitor cell (HSPC) formation during embryoid body differentiation (Doulatov et,al., 2013); and (3) Foxc1, which is highly expressed the CAR cells, a critical cell type for HSC maintenance (Oomatsu et al.,2014). We injected iPSCs into recipient mice, without co-injection of stromal cells or cytokines, and induced TF expression after teratoma formation by dox administration. After four weeks, GFG-derived teratomas contained large numbers of endothelial-like and epithelial-like cells, and importantly GFG-derived hematopoietic cells could also be detected. EAR-teratomas also generated hematopoietic cells, although at lower frequencies. By contrast, hematopoietic cells were not detected in control teratomas or Foxc1-teratomas. Through use of iPSCs generated from Runx1-EGFP mice (Ng et al. 2010), and CUBIC 3D imaging technology (Susaki et al. 2014), we were further able to demonstrate that GFG-derived hematopoietic cells were generated through a haemogenic endothelium precursor. Next, we assessed whether HSPC-deficient recipient mice would allow greater expansion of teratoma-derived HSCs. This was achieved by inducing c-kit deletion within the hematopoietic compartment of recipient mice (Kimura et al., 2011) and resulted in a ten-fold increase in the peripheral blood frequency of iPSC-derived hematopoietic cells. We further confirmed similar increases in iPSC-derived bone marrow cells, and in vivo HSC expansion, through bone marrow transplantation assays. Finally, we have been able to shorten the HSC generation time in this assay by five weeks through use of transplantable teratomas, rather than iPSCs. Conclusions We have demonstrated that GFG-iPSCs induce HSC generation within teratomas, via a hemogenic endothelium precursor, and that use of HSPC-deficient recipient mice further promotes expansion of teratoma-derived HSCs. These modifications now allow us to generate engraftable HSCs without co-injection of stromal cells or cytokines. Additionally, use of transplantable teratomas reduced HSC generation times as compared with the conventional assay. These findings suggest that our in vivo system provides a promising strategy to generate engraftable HSCs from iPSCs. Disclosures No relevant conflicts of interest to declare.

Interactions between human adipose stromal cells and mouse neural stem cells in vitro

2003 ◽  
Vol 145 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Soo Kyung Kang ◽  
Eun Sook Jun ◽  
Yong Chan Bae ◽  
Jin Sup Jung
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Stromal Cells ◽  
Adipose Stromal Cells

scholarly journals Myotubes derived from human-induced pluripotent stem cells mirror in vivo insulin resistance

2016 ◽  
Vol 113 (7) ◽  
pp. 1889-1894 ◽  
Author(s):  
Salvatore Iovino ◽  
Alison M. Burkart ◽  
Laura Warren ◽  
Mary Elizabeth Patti ◽  
C. Ronald Kahn
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ips Cells ◽  
Muscle Insulin Resistance ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPS cells) represent a unique tool for the study of the pathophysiology of human disease, because these cells can be differentiated into multiple cell types in vitro and used to generate patient- and tissue-specific disease models. Given the critical role for skeletal muscle insulin resistance in whole-body glucose metabolism and type 2 diabetes, we have created a novel cellular model of human muscle insulin resistance by differentiating iPS cells from individuals with mutations in the insulin receptor (IR-Mut) into functional myotubes and characterizing their response to insulin in comparison with controls. Morphologically, IR-Mut cells differentiated normally, but had delayed expression of some muscle differentiation-related genes. Most importantly, whereas control iPS-derived myotubes exhibited in vitro responses similar to primary differentiated human myoblasts, IR-Mut myotubes demonstrated severe impairment in insulin signaling and insulin-stimulated 2-deoxyglucose uptake and glycogen synthesis. Transcriptional regulation was also perturbed in IR-Mut myotubes with reduced insulin-stimulated expression of metabolic and early growth response genes. Thus, iPS-derived myotubes from individuals with genetically determined insulin resistance demonstrate many of the defects observed in vivo in insulin-resistant skeletal muscle and provide a new model to analyze the molecular impact of muscle insulin resistance.

scholarly journals Lithium stimulation of murine hematopoiesis in liquid culture: an effect mediated by marrow stromal cells

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 121-127 ◽  
Author(s):  
PJ Quesenberry ◽  
MA Coppola ◽  
RJ Gualtieri ◽  
PM Wade ◽  
Z Song ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Two Phase ◽  
Support Cell ◽  
Macrophage Colony ◽  
Culture Growth ◽  
Stimulation Of

Abstract Lithium has previously been observed to stimulate in vitro Dexter culture hemopoiesis with increases in granulocytes, megakaryocytes, and pluripotent stem cells (CFU-S). In the present study, a two-phase murine Dexter culture system was established to study the mechanism of lithium-mediated stem cell stimulation. Different lots of horse sera or fetal calf sera were found to have markedly different effects on Dexter culture growth; given the appropriate sera supplementation, supernatant cells from Dexter cultures established from C57BL/6J mice 3 wk previously were free of stromal-forming capacity, but had stem cells and could grow on 900–950 R irradiated stroma. Conversely, in vitro irradiation (900–950 R) of 3-wk cultures resulted in a stem-cell-free adherent monolayer that could support growth for up to 9 wk in culture. The stroma from Dexter cultures preexposed to lithium chloride (1.0 mmole/liter) for 3 wk, irradiated (900 R), and then refed with 3-wk Dexter supernatant cells has an enhanced capacity to support cell production, CFU-S, and probably granulocyte-macrophage colony-forming cell (GM-CFU-C) production, as compared to stroma not preexposed to lithium. Lithium carryover was ruled out in these experiments. These data indicate that lithium stimulates CFU-S and in vitro granulopoiesis by an indirect effect on a radioresistant adherent stromal cell.

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