192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS

2014 ◽  
Vol 26 (1) ◽  
pp. 210
Author(s):  
M. Nowak-Imialek ◽  
N. Lachmann ◽  
D. Herrmann ◽  
F. Jacob ◽  
H. Niemann
Keyword(s):  
Stem Cells ◽  
Cell Population ◽  
Germ Cells ◽  
Germ Line ◽  
Cell Mass ◽  
Testicular Tissue ◽  
Egfp Expression ◽  
Cell Populations ◽  
Marker Genes ◽  
Transgenic Pigs

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.

324 TESTIS-SPECIFIC EXPRESSION OF Oct4-EGFP TRANSGENE IN PIG

2015 ◽  
Vol 27 (1) ◽  
pp. 251
Author(s):  
M. Nowak-Imialek ◽  
N. Lachmann ◽  
D. Herrmann ◽  
F. Jacob ◽  
H. Niemann
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Leydig Cells ◽  
Egfp Expression ◽  
Seminiferous Tubules ◽  
Marker Genes ◽  
Specific Expression ◽  
Oct4 Expression ◽  
Egfp Reporter

Oct4 is a transcription factor essential for establishment and maintenance of pluripotency in mammalian stem cells. Oct4 expression was found in early embryos and germ cells throughout fetal development. In male mice, Oct4 expression is found in mitotically arrested prospermatogonia until birth. After onset of spermatogenesis, expression is maintained in type A spermatogonia, but is downregulated in type B spermatogonia and in spermatocytes (Pesce et al. 1998 Mech. Dev). Previously, we successfully generated Oct4-EGFP reporter pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev.). This animal model is unique because it allows in vivo and in vitro visualisation of Oct4-positive cells. Germ line specific Oct4-EGFP expression was analysed in testis isolated from young (<1 week) and adult (>7 months) pigs. Squash preparation of testicular tissue isolated from adult transgenic boars revealed high amounts of EGFP-positive cells compared to young piglets. We confirmed Oct4 and EGFP expression in the testis from young and adult transgenic animals using Northern blot analysis. Specific expression of Oct4 and EGFP in testis could be observed in blots as a single band of 1.5 kb. As a loading control, the blot was rehybridized with a β-actin probe. Mammalian testes contain different cell types, including germ cells, Sertoli cells, Leydig cells, and peritubular cells. To define the cellular origin of EGFP-expressing cells, we isolated these cells from adult transgenic testis using fluorescence-activated cell sorting (FACS)-based techniques. Analysis of isolated EGFP positive cells with qRT-PCR demonstrated the presence of marker genes specific for undifferentiated (Oct4, UTF1, FGFR3, PGP 9.5, THY-1, SALL4, and GFRα1) and differentiated (BOLL and PRM2) germ cells. Markers specific for Sertoli cells (vimentin) and Leydig cells (LHCGR) were not observed. To verify the localization of EGFP-positive cells in seminiferous tubules, we performed immunohistochemical detection of GFP in adult pig testis. Unlike the Oct4-EGFP reporter mouse model, GFP protein was not found in spermatogonia attached to the basement membrane of seminiferous tubules, but instead were found in differentiated germ cells, including spermatocytes and spermatids. These results show that the Oct4-EGFP expression in testis differs between mouse and porcine Oct4-EGFP transgenic models. To verify that the EGFP expression driven by the mouse Oct4 promoter in porcine testis reflects the endogenous Oct4 expression profile, Western blot and histochemical analyses are currently underway.

286 EXPRESSION OF PLURIPOTENT STEM CELL MARKERS IN DOMESTIC CAT SPERMATOGONIAL CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 290 ◽  
Author(s):  
R. H. Powell ◽  
M. N. Biancardi ◽  
J. Galiguis ◽  
Q. Qin ◽  
C. E. Pope ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Basement Membrane ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Surface Markers ◽  
Cell Markers

Spermatogonial stem cells (SSC), progenitor cells capable of both self-renewal and producing daughter cells that will differentiate into sperm, can be manipulated for transplantation to propagate genetically important males. This application was demonstrated in felids by the successful xeno-transplantation of ocelot mixed germ cells into the testes of domestic cats, which resulted in the production of ocelot sperm (Silva et al. 2012 J. Androl. 33, 264–276). Spermatogonial stem cells are in low numbers in the testis, but have been identified and isolated in different mammalian species using SSC surface markers; however, their expression varies among species. Until recently, little was known about the expression of SSC surface markers in feline species. We previously demonstrated that many mixed germ cells collected from adult cat testes express the germ cell markers GFRα1, GPR125, and C-Kit, and a smaller population of cells expresses the pluripotent SSC-specific markers SSEA-1 and SSEA-4 (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222). In the present study, our goal was to identify germ cell and SSC-specific markers in SSC from cat testes. Immunohistochemical (IHC) localization of germ cell markers GFRα1, GPR125, and C-Kit and pluripotent SSC-specific markers SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 was detected in testis tissue from both sexually mature and prepubertal males. Testes were fixed with modified Davidson’s fixative for 24 h before processing, embedding, and sectioning. The EXPOSE Mouse and Rabbit Specific HRP/DAB detection IHC kit (Abcam®, Cambridge, MA, USA) was used for antibody detection. Staining for SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 markers was expressed specifically at the basement membrane of the seminiferous tubules in both adult and prepubertal testes. The GFRα1 and GPR125 markers were detected at the basement membrane of the seminiferous tubules and across the seminiferous tubule section. However, C-Kit was not detected in any cell. Using flow cytometry from a pool of cells from seven adult testes, we detected 45% GFRα1, 50% GPR125, 59% C-Kit, 18% TRA-1-60, 16% TRA-1-81 positive cells, and a very small portion of SSEA-1 (7%) and SSEA-4 (3%) positive cells. Dual staining of germ cells pooled from 3 testes revealed 3 distinct cell populations that were positive for GFRα1 only (23%), positive for both GFRα1 and SSEA-4 (6%), and positive for SSEA-4 only (1%). Our IHC staining of cat testes indicated that cells along the basement membrane of seminiferous tubules were positive for SSC-specific markers, and flow cytometry analysis revealed that there were different cell populations expressing both germ cell and SSC-specific markers. Flow cytometry results show overlapping germ cell populations expressing SSEA-4 and GFRα1, and IHC results reveal that SSEA-4 positive cells are spermatogonia, whereas GFRα1 positive cells include other stages of germ cells, indicating that the small population of cells positive only for SSEA-4 is undifferentiated cat SSC.

Male germ line stem cells: from cell biology to cell therapy

2003 ◽  
Vol 15 (6) ◽  
pp. 323 ◽  
Author(s):  
David Pei-Cheng Lin ◽  
Ming-Yu Chang ◽  
Bo-Yie Chen ◽  
Han-Hsin Chang
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Germ Line ◽  
Current Status ◽  
Seminiferous Tubules ◽  
Male Germ Line ◽  
Male Germ Cells ◽  
Stem Cell Lines

Research using stem cells has several applications in basic biology and clinical medicine. Recent advances in the establishment of male germ line stem cells provided researchers with the ability to identify, isolate, maintain, expand and differentiate the spermatogonia, the primitive male germ cells, as cell lines under in vitro conditions. The ability to culture and manipulate stem cell lines from male germ cells has gradually facilitated research into spermatogenesis and male infertility, to an extent beyond that facilitated by the use of somatic stem cells. After the introduction of exogenous genes, the spermatogonial cells can be transplanted into the seminiferous tubules of recipients, where the transplanted cells can contribute to the offspring. The present review concentrates on the origin, life cycle and establishment of stem cell lines from male germ cells, as well as the current status of transplantation techniques and the application of spermatogonial stem cell lines.

scholarly journals Glioblastoma embryonic-like stem cells exhibit immune-evasive phenotype

2021 ◽  
Author(s):  
Borja Sese ◽  
Sandra Iniguez ◽  
Miquel Arash Ensenat ◽  
Pere Llinas ◽  
Guillem Ramis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Immune Evasion ◽  
Cell Mass ◽  
Induced Pluripotent Stem Cell ◽  
Stem Cell Population ◽  
Glioma Stem Cells ◽  
Pluripotency Markers

Glioma stem cells (GSCs) are a subset of cells with self-renewal and tumor-initiating capacities that are thought to participate in drug resistance and immune evasion mechanisms in glioblastoma (GBM). Given GBM heterogeneity, we hypothesized that GSCs might also display cellular hierarchies associated with different degrees of stemness. We evaluated a single-cell RNA-seq glioblastoma dataset (n = 28) and identified a stem cell population co-expressing high levels of embryonic pluripotency markers, named core glioma stem cells (c-GSCs). This embryonic-like population represents 4.22% of the tumor cell mass, and pathway analysis revealed an upregulation of stemness and downregulation of immune-associated pathways. Using induced pluripotent stem cell technology, we generated an in vitro model of c-GSCs by reprogramming glioblastoma patient-derived cells into induced c-GSCs (ic-GSCs). Immunostaining of ic-GSCs showed high expression of embryonic pluripotency markers and downregulation of antigen presentation HLA proteins, mimicking its tumoral counterpart. Transcriptomic analysis revealed a strong agreement of enriched biological pathways between tumor c-GSCs and in vitro ic-GSCs (k = 0.71). Integration of ic-GSC DNA methylation and gene expression with chromatin state analysis of epigenomic maps (n = 833) indicated that polycomb repressive marks downregulate HLA genes in stem-like phenotype. Together, we identified c-GSCs as a GBM cell population with embryonic signatures and poor immunogenicity. Genome-scale transcriptomic and epigenomic profiling provide a valuable resource for studying immune evasion mechanisms governing c-GSCs and identifying potential therapeutic targets for GBM immunotherapy.

scholarly journals Myeloperoxidase gene expression in blast cells with a lymphoid phenotype in cases of acute lymphoblastic leukemia

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 873-876 ◽  
Author(s):  
S Ferrari ◽  
MT Mariano ◽  
E Tagliafico ◽  
M Sarti ◽  
G Ceccherelli ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Population ◽  
Blot Analysis ◽  
Germ Line ◽  
Lymphoblastic Leukemia ◽  
Cell Populations ◽  
Chain Gene ◽  
Blast Cells ◽  
Chain Gene Rearrangement ◽  
Ig Heavy Chain

By using a cDNA clone of the myeloperoxidase (MPO) gene, we have studied, by Northern blot analysis, the level of MPO mRNA in eight cases of acute lymphoblastic leukemia (ALL). The blast cell populations studied were characterized by morphologic, cytochemical, immunochemical, and molecular criteria. With all the methods used the populations were found to be highly homogeneous and showed a typical lymphoid phenotype. In particular, the Ig heavy-chain gene rearrangement was largely prevalent, and the germ line configuration was almost absent. However, in three of eight cases, high levels of MPO mRNA were detected. The remarkable homogeneity of the cell populations examined suggests that the MPO mRNA observed was present in cellular elements certainly identified as lymphoid. The absence of contamination by myeloid cells was confirmed by the results of Western blot analysis of the proteins of the cell population studied: no MPO protein was detectable. The levels of mRNA observed were high enough to be comparable to those observed in a promyelocytic cell population.

The development of the testis of the Merino ram, with special reference to the orgin of the adult stem cells

1962 ◽  
Vol 13 (3) ◽  
pp. 487 ◽  
Author(s):  
CS Sapsford
Keyword(s):  
Stem Cells ◽  
Basement Membrane ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Adult Stem Cells ◽  
Germ Line ◽  
Primordial Germ Cells ◽  
Seminiferous Tubules ◽  
Stages Of Development ◽  
Striking Change

In the ram, as in other mammals, the sex cords are made up of two types of cell: indifferent cells (derivatives of the coelomic epithelium) and primordial germ cells. In the cords, each type pursues a separate and independent line of development to become respectively the Sertoli cells and the stem cells (type A spermatogonia) of the adult testis. The principal changes taking place in the primordial germ cells (gonocytes) are a reduction in the size and number of the Feulgen-positive particles in the nuclei, the appearance and subsequent fusion of the nucleoli, and, finally, an increase in the size of the nuclei. While these changes are taking place, the cytoplasm increases in volume and inclusions become more numerous. Cells which have undergone all these transformations have been called prospermatogonia. The cells of the germ line are at first more centrally placed in the sex cords than the indifferent cells. Just before spermatogenesis begins, they migrate to the basement membrane of the seminiferous tubules. All germ cells in tubules in which spermatogenesis has been initiated are seen as prospermatogonia. These cells become flattened against the basement membrane, and their nuclei become more oval in shape. They thus become identical with the stem cells of the adult. Little change is evident in the nuclei of the indifferent cells until puberty. Feulgen-positive material is found in the form of coarse granules at earlier stages of development. At puberty, these granules become dispersed to give a much more homogeneous nucleus. Concurrently, nuclei increase in size, and single or double true nucleoli can be identified. During development, increases in cytoplasmic volume take place. Although cell boundaries between indifferent cells cannot be seen in fixed material, phase contrast observations of fresh material have demonstrated that some forms exist as mononucleate units. It could not be determined whether the same was true in the case of Sertoli cells. No striking change in the relative numbers of glandular interstitial cells could be observed at different stages of development.

429 GENERATION OF Oct4-EGFP TRANSGENIC PIGS FOR MONITORING REPROGRAMMING AND PLURIPOTENCY

2010 ◽  
Vol 22 (1) ◽  
pp. 371 ◽  
Author(s):  
M. Nowak-Imialek ◽  
W. A. Kues ◽  
B. Petersen ◽  
A. Lucas-Hahn ◽  
D. Herrmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Embryonic Stem ◽  
Large Animal Model ◽  
Large Animal ◽  
Transgenic Pigs ◽  
Egfp Fluorescence

The Oct4 gene is an essential transcription factor for maintenance of pluripotency in mammals. Here, we report the production of cloned transgenic pigs carrying a genomic construct encompassing murine Oct4 regulatory regions and driving an enhanced green fluorescent protein (Oct4-EGFP) construct. We employed fetal porcine fibroblasts, stably co-transfected with neomycin and the mouse Oct4-EGFP construct, for somatic cell nuclear transfer to reconstruct transgenic embryos. The cloned embryos (811 embryos) were surgically transferred into the oviducts of 8 recipient animals. Two pregnancies were terminated at Day 25 for recovery of fetuses and the others delivered a total of 23 piglets, of which 11 survived the postpartum period. A detailed analysis showed that the Oct4-EGFP construct was active in cloned pig blastocysts from Days 5 to 6. EGFP fluorescence was found exclusively in the primordial germ cells of Day 25 fetuses, whereas somatic tissues did not express the transgene. We could also detect expression of Oct4-EGFP in individual cells of the postnatal testis. Testis-specific expression was confirmed by Northern blotting. We fused transgenic porcine fibroblasts with murine embryonic stem cells to analyze reactivation of the Oct4-EGFP transgene under experimental reprogramming conditions. The fused hybrids displayed stem cell morphology and a high proliferation rate and started to express EGFP fluorescence 72 h after fusion. In conclusion, we report the production of viable Oct4-EGFP transgenic piglets that express EGFP exclusively in germ line and pluripotent cells. This transgenic pig line is a valuable tool for derivation and maintenance of porcine embryonic stem cells and will be of utmost interest for reprogramming studies and for preclinical testing of stem cell therapies in a large animal model. Funded by BMBF.

CD133 Marks a Stem Cell Population That Drives Human Primary Myelofibrosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1601-1601
Author(s):  
Ioanna Triviai ◽  
Thomas Stuebig ◽  
Birte Niebuhr ◽  
Kais Hussein ◽  
Asterios Tsiftsoglou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Myeloid Cell ◽  
Primary Myelofibrosis ◽  
Jak2v617f Mutation ◽  
Stem Cell Population ◽  
Cell Populations ◽  
Cd34 Cells

Abstract Primary Myelofibrosis (PMF) is a chronic myeloproliferative neoplasm of alleged stem cell origin. To define the characteristics of malignant PMF stem cells previous studies have focused on the isolation and xenotransplantation of circulating and/or splenic, PMF patient - derived CD34+ stem/progenitor cells. Despite the reported engraftment of CD34+ cell pool, former analyses failed to reproduce major PMF parameters attributed to abnormal human myeloid cell differentiation. The focus of our work was to identify the stem cell population responsible for initiation and development of PMF. To assess the presence of malignant stem cells we analyzed peripheral blood of 30 PMF patients for expression of LT-HSC antigen CD133. To exclude committed myeloid and lymphoid circulating progenitors we performed lineage depletion of PBMCs and isolated CD133+ and/or CD34+ stem cells. Variable CD133+/CD34 ± and CD133-/CD34+ stem cell fractions from 15 PMF patients were assessed for their clonogenic potential in semisolid media and for reproduction of PMF morbidity in a xenotransplantation mouse model. JAK2V617F mutation was used as a genetic marker to track clonal evolution both in vitro and in vivo. In patients' PBMC we detected the consistent presence of a CD133+ population ranging from 0.3% to >30%, which varies in the expression of CD34. CD133 marks overlapping but also distinct cell populations as compared to CD34. To determine the differentiation potential of disparate stem cell populations, CD133+CD34+, CD133-CD34+ and CD133+CD34- cells were subfractionated from PB of 7 patients and assessed for clonogenic capacity. Strikingly, CD133+CD34+ cells exhibited multipotent, bipotent, and unipotent myeloid (including erythroid) and endothelial-like output, whereas CD133-CD34+ cells gave rise predominantly to lineage-restricted granulocyte/monocyte (GM) progenitors or endothelial-like progenitors. Thus, in contrast to circulating CD133-/CD34+ cells in PMF patients, CD133+ cells have a broader and more robust differentiation capacity to all myeloid cell types, including megakaryocyte /erythrocyte lineages. Four JAK2V617F+ patient samples were used to assess mutation burden at the single-cell level from representative colony types. Obtained results demonstrate an early acquisition of JAK2V617F mutation in the primitive CD133+ stem cell compartments, but also revealed an unexpected variability in the genotypes of emerging progenitors. Homozygous JAK2617F/617F progenitors were detectable in all analyzed patient samples, even if a relative low JAK2V617F burden (30%) was determined from the initial pool of CD133+ cells. A disproportionately high incidence of a homozygous JAK2V617F genotype was observed in erythroid progenitors, indicating a skewing for this lineage. Homozygosity was additionally detected in megakaryocytic and multipotent progenitors. In vivo xenotransplantation experiments of various subfractions confirm the origin of multipotent JAK2V617F+ progenitors from CD133+/CD34± stem cells. Transplantation of PMF patient-derived CD133+/CD34± stem cells in immuno-compromised mice induces abnormal human JAK2V617F+ erythroid, megakaryocytic, and monocytic differentiation, splenomegaly, bone marrow/splenic fibrosis and anemia, reproducing many aspects of PMF development. Our data provide the first evidence for the existence of a CD133+ LT-HSC population responsible for development of PMF. It is for the first time demonstrated that JAK2V617F mutation in PMF occurs at the level of a multipotent stem cell, from which all abnormal myeloid cells emanate during evolution of the disease. Identification of the stem cell compartment involved in the triggering and progression of PMF provides the basis to elucidate the nature of the complex niche interactions in myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Scaffold-Free Fabrication of Osteoinductive Cellular Constructs Using Mouse Gingiva-Derived Induced Pluripotent Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Hiroko Okawa ◽  
Hiroki Kayashima ◽  
Jun-Ichi Sasaki ◽  
Jiro Miura ◽  
Yuya Kamano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cell Mass ◽  
Embryoid Bodies ◽  
Marker Genes ◽  
Induction Medium ◽  
Gingival Fibroblast ◽  
Osseous Tissue ◽  
Induced Pluripotent

Three-dimensional (3D) cell constructs are expected to provide osteoinductive materials to develop cell-based therapies for bone regeneration. The proliferation and spontaneous aggregation capability of induced pluripotent stem cells (iPSCs) thus prompted us to fabricate a scaffold-free iPSC construct as a transplantation vehicle. Embryoid bodies of mouse gingival fibroblast-derived iPSCs (GF-iPSCs) were seeded in a cell chamber with a round-bottom well made of a thermoresponsive hydrogel. Collected ball-like cell constructs were cultured in osteogenic induction medium for 30 days with gentle shaking, resulting in significant upregulation of osteogenic marker genes. The constructs consisted of an inner region of unstructured cell mass and an outer osseous tissue region that was surrounded by osteoblast progenitor-like cells. The outer osseous tissue was robustly calcified with elemental calcium and phosphorous as well as hydroxyapatite. Subcutaneous transplantation of the GF-iPSC constructs into immunodeficient mice contributed to extensive ectopic bone formation surrounded by teratoma tissue. These results suggest that mouse GF-iPSCs could facilitate the fabrication of osteoinductive scaffold-free 3D cell constructs, in which the calcified regions and surrounding osteoblasts may function as scaffolds and drivers of osteoinduction, respectively. With incorporation of technologies to inhibit teratoma formation, this system could provide a promising strategy for bone regenerative therapies.

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