scholarly journals In vitro studies of human pluripotential hematopoietic progenitors in polycythemia vera. Direct evidence of stem cell involvement.

1982 ◽  
Vol 69 (5) ◽  
pp. 1112-1118 ◽  
Author(s):  
R C Ash ◽  
R A Detrick ◽  
E D Zanjani
Keyword(s):  
Stem Cell ◽  
Polycythemia Vera ◽  
Direct Evidence ◽  
In Vitro Studies ◽  
Hematopoietic Progenitors

scholarly journals Heparanase regulates retention and proliferation of primitive Sca-1+/c-Kit+/Lin− cells via modulation of the bone marrow microenvironment

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 4934-4943 ◽  
Author(s):  
Asaf Spiegel ◽  
Eyal Zcharia ◽  
Yaron Vagima ◽  
Tomer Itkin ◽  
Alexander Kalinkovich ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Cathepsin K ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Cell Mobilization ◽  
Tumor Growth And Metastasis

Abstract Heparanase is involved in tumor growth and metastasis. Because of its unique cleavage of heparan sulfate, which binds cytokines, chemokines and proteases, we hypothesized that heparanase is also involved in regulation of early stages of hematopoiesis. We report reduced numbers of maturing leukocytes but elevated levels of undifferentiated Sca-1+/c-Kit+/Lin− cells in the bone marrow (BM) of mice overexpressing heparanase (hpa-Tg). This resulted from increased proliferation and retention of the primitive cells in the BM microenvironment, manifested in increased SDF-1 turnover. Furthermore, heparanase overexpression in mice was accompanied by reduced protease activity of MMP-9, elastase, and cathepsin K, which regulate stem and progenitor cell mobilization. Moreover, increased retention of the progenitor cells also resulted from up-regulated levels of stem cell factor (SCF) in the BM, in particular in the stem cell–rich endosteum and endothelial regions. Increased SCF-induced adhesion of primitive Sca-1+/c-Kit+/Lin− cells to osteoblasts was also the result of elevation of the receptor c-Kit. Regulation of these phenomena is mediated by hyperphosphorylation of c-Myc in hematopoietic progenitors of hpa-Tg mice or after exogenous heparanase addition to wildtype BM cells in vitro. Altogether, our data suggest that heparanase modification of the BM microenvironment regulates the retention and proliferation of hematopoietic progenitor cells.

Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4641-4651 ◽  
Author(s):  
Pankaj Gupta ◽  
Theodore R. Oegema ◽  
Joseph J. Brazil ◽  
Arkadiusz Z. Dudek ◽  
Arne Slungaard ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Stem Cell Niche ◽  
Ex Vivo ◽  
Recent Observation ◽  
Hematopoietic Progenitors ◽  
Platelet Factor ◽  
Cell Niche

Abstract Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P < .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

Negative Control Region Is a Critical Element Of Insertional Oncogenesis After Gene Transfer Into Hematopoietic Progenitors With Moloney Murine Leukemia Viruses

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 164-164
Author(s):  
Yasuhiro Ikawa ◽  
Toru Uchiyama ◽  
Guridevi Jayashree Jagadeesh ◽  
Fabio Candotti
Keyword(s):  
Stem Cell ◽  
Gene Transfer ◽  
Viral Vectors ◽  
Negative Control ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Cell Immortalization ◽  
The Difference ◽  
Murine Leukemia

Abstract Gene transfer into hematopoietic stem cells has been used successfully to treat a variety of human genetic diseases. Although protocols have shown positive clinical outcomes, the successes of clinical trials have been tempered by adverse events in which the use of gamma-retroviral vectors (GV) containing full-length long terminal repeats (LTRs) with strong enhancer activity increased transcription of cancer-related genes, and thereby contributed to development of leukemia. Assessing safety of integrating viral vectors for future clinical use is therefore of paramount importance. The negative control region (NCR) is a particularly well-conserved sequence among mammalian gamma-retroviruses with demonstrated regulating a transcription activity of GV in hematopoietic cells. This suggests that the NCR might play a crucial role of insertional oncogenesis after gene transfer into hematopoietic progenitors. In a series of safety studies of viral gene transfer constructs, we used an in vitro assay of murine bone marrow (BM) cell immortalization and compared the consequences of hematopoietic stem cell transduction with three different kinds of viral vectors, including Moloney murine leukemia virus- (MMLV), lentivirus- (LV), and foamy virus (FV)-based constructs. To evaluate critical elements for cell immortalization by MMLV vectors, we also tested four different MMLV LTR variants deleted of either 1) most of the two 75-bp repeats associated with the viral enhancer (delE1), 2) all of the two 75-bp repeats and the NCR (delE2), 3) only the NCR (delNCR), or 4) carrying a deleterious mutation of the NCR NFAT motif (ΔNFAT). All vectors carried an internal expression cassette including the eGFP gene under the control of a UCOE (ubiquitously acting chromatin opening element) promoter. In this assay, BM cells are harvested from C57BL6 mice, exposed to retroviral supernatants and cultured long-term. Derived lines are considered immortalized based on their ability to continue to grow in vitro for more than six weeks in the presence of interleukin-3 and stem cell factor. Real-time PCR was performed to verify comparable transduction efficiency of bone marrow cells by different vectors. In our analysis of MMLV LTR mutants, full-MMLV and delE1 transduction of 92 and 108 cultures, respectively, resulted in 37 and 37 immortalized lines (40% and 34% immortalization rate, respectively). The difference in immortalization rate between full-MMLV and delE1 was not statistically significant. Transductions using delE2-, delNCR- and ΔNFAT-carrying vectors of 60, 36 and 35 cultures resulted in 10, 3 and 10 immortalized lines (17%, 8.3% and 29% immortalization rate, respectively). The difference between the immortalization caused by delE1 and delE2 vectors was statistically significant (p<0.05). Moreover, the difference between the immortalization caused by full-MMLV and delNCR vectors was statistically significant (p<0.01), while there was no significant difference between the immortalization induced by full-MMLV and ΔNFAT vectors. Transduction of 57 and 34 cultures with LV and FV vectors, respectively, resulted in no immortalized lines. Transductions of 128 cultures with a LV construct carrying the U3 region from the murine stem cell virus LTR as an internal promoter (LV-U3) resulted in 2 immortalized lines which was not statistically different from the results obtained with LV vectors carrying the UCOE internal promoter. These results confirm that GV are prone to causing immortalization of hematopoietic cells and indicate that deletion of the whole viral enhancer sequences may not be adequate to eliminate the insertional oncogenesis risk. Importantly, our data point to the NCR as a crucial element for immortalization and justify additional studies to evaluate its specific role in MMLV-mediated insertional oncogenesis. Finally, our results suggest that vectors based on LV and FV backbones are safer alternatives for clinical gene transfer into hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of Mesenchymal Stem Cell-Conditioned Medium (MSC-CM) in the Bone Regeneration: A Systematic Review from 2007-2018

2019 ◽  
pp. 1-16
Author(s):  
Ismail Hadisoebroto Dilogo ◽  
Jessica Fiolin
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Bone Regeneration ◽  
Conditioned Medium ◽  
Bone Healing ◽  
In Vitro Studies ◽  
In Vivo Studies

Background: The therapeutic value of mesenchymal stem cells (MSCs) in tissue engineering and regenerative medicine is attributable in part to paracrine pathways triggered by several secreted factors secreted into culture media. The secreted factor here is known as the conditioned medium (CM) or secretome. Objectives: This review is aimed to investigate and summarise the in-vitro, pre-clinical in-vivo studies regarding the role of CM-MSC in bone regeneration from 2007 until 2018 Data Sources: A systematic literature search on PubMed, MEDLINE, OVID, Scopus and Cochrane library was carried out by using search terms: Secretome, conditioned medium, mesenchymal stem cell, bone healing, osteogenic, osteogenesis. Methods: A total of 611 articles were reviewed. Ten articles were identified as relevant for this systematic literature review. Results: Three tables of studies were constructed for in vitro studies and in-vivo studies. Conclusion: All of the included in-vitro studies and in-vivo studies have shown a promoting effect of bone regeneration at various stages. Although there are no clinical studies regarding the use of CM-MSC in the human bone regeneration that have been conducted, transplantation of secretome has shown a promising result in the acceleration of bone healing process.

scholarly journals Analysis of Leukemogenic Effects of RUNX1 and CSF3R Mutations Using Congenital Neutropenia (CN)/AML Patient-Derived Induced Pluripotent Stem Cells (iPSCs)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 404-404
Author(s):  
Benjamin Dannenmann ◽  
Maksim Klimiankou ◽  
Christian Lindner ◽  
Azadeh Zahabi ◽  
Regine Bernhard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Trisomy 21 ◽  
Bone Marrow Failure ◽  
Hematopoietic Cells ◽  
Hematopoietic Progenitors ◽  
Congenital Neutropenia ◽  
Induced Pluripotent

Abstract Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. Recently we reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. Only a combination of these two mutations induced elevated proliferation and diminished myeloid differentiation of CD34+ cells in vitro. To confirm these clinical data in an in vitro model, we generated human induced pluripotent stem cells (hiPSCs) from PBMNCs of a CN patient harbouring p.C151Y mutation in ELANE after acquisition of AML. During GCSF treatment, this patient acquired G-CSFRmutation p.Q741*, which leads to a truncated G-CSF receptor and was detected six years prior to overt AML. Three years later, he acquired an additional RUNX1 (p.R139G) mutation, which is located in the RUNT-homology domain (RHD). Subsequently, he developed AML (FAB M1) with trisomy 21. Reprogramming of PBMNCs isolated from the time-point of AML (ca. 80 % of AML blasts) resulted in the generation of hiPSCs clones harbouring either only ELANE p.C151Y mutation (CN-iPSC clone, derived from non-leukemia PBMNCs) or additional CSF3R and RUNX1 mutations and trisomy 21 (CN/AML-iPSC clone, derived from AML blasts), which was subsequently validated by Sanger sequencing and by digital PCR. These iPSCs clones have been tested for their pluripotency and self-renewal capacity. Both iPSC clones expressed the pluripotent stem cell surface markers SSEA-4 and TRA-1-60 and displayed alkaline phosphatase activity. Further they highly expressed mRNA of the pluripotent stem cell markers SOX2, ABCG2, DNMT and NANOG and were able to differentiate into all three germ layers (meso-, endo- and ectoderm). Embryoid body (EB)-based hematopoietic / neutrophilic differentiation of CN-iPS clones using serum-free APEL stem cell differentiation medium showed comparable amounts of CD34+ and CD33+ cells, but ~ 2-fold reduction of CD16+ cells, compared to healthy donor (HD) iPSCs. CN/AML-iPSCs were not able to differentiate into mature granulocytes at all and revealed 10-fold reduced counts of CD34+ and CD33+hematopoietic cells. Morphological examinations of Giemsa-stained cytospin slides confirmed these results. Additionally, CN/AML-iPSCs showed a highly reduced number of CFU-G and CFU-GM colonies in CFU-Assay. To investigate the intracellular mechanisms of leukemogenic transformation in CN, we analyzed gene expression profiles of hematopoietic cells generated from CN-iPSCs vs CN/AML-iPSCs and HD-iPSCs for various time points of differentiation in our EB based-system. Our previous microarray-based analysis of bone marrow CD33+ cells of this CN/AML patient revealed that genes overexpressed in early hematopoietic stem/progenitor cells (HSPCs) as compared to more mature progenitors, such as DNTT, BAALC, CD34, HPGDS, NPR3 and PROM1 were strongly upregulated in CN/AML blasts harbouring both RUNX1 and CSF3R mutations, as compared to the cells prior to leukemia development. Intriguingly, elevated expression of these genes was described previously in RUNX1-mutated de novo AML blasts (Mendler et al., JCO 2012). This genetic signature suggests transformation of hematopoietic progenitors carrying mutated CSF3R into more primitive hematopoietic progenitors after aquisition of RUNX1mutation. We were able to confirm markedly increase of mRNA levels of these genes in hematopoietic cells derived from CN/AML-iPSCs, as compared to CN-iPSCs. In addition, we found that hematopoietic cells of both CN-iPSCs and CN/AML-iPSCs revealed increased expression of unfolded-protein response (UPR) genes DDIT3 (CHOP), ATF4 and ATF6, as compared to HD-iPSCs. Activation of UPR in hematopoietic cells of CN-ELANEpatients has been previously described by our and other groups. CN/AML-iPSC-derived hematopoietic progenitor cells expressed RUNX1 mRNA at least two-fold higher, as compared to HD- or CN-iPSC-derived cells. In summary, we established an in vitro cellular model of leukemogenic transformation in CN patients using CN/AML-patient derived hiPSCs that confirmed clinical data of Skokowa et al. (Blood 123:2550, 2014) on a cooperative leukemogenic effect of CSF3R and RUNX1 mutations. Comprehensive analysis of hematopoiesis using this iPSCs model will give us a deeper view into this highly complex signaling network operating during leukemogenic transformation of HSCs in pre-leukemic bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2045-2053 ◽  
Author(s):  
CM Verfaillie
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Expansion ◽  
Terminal Differentiation ◽  
Hematopoietic Progenitors ◽  
Soluble Factors ◽  
Bone Marrow Cultures ◽  
Stromal Layer

We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors, we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage- /CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer (“stroma-free”) or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane (“stroma-noncontact”). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a “stroma-free” culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3], stem cell factor [SCF], leukemia-inhibitory factor [LIF], and granulocyte colony-stimulating factor [G-CSF]) results in a similar cell expansion as when DR- cells are cultured in “stroma-noncontact” cultures supplemented with the same cytokines. However, generation of committed progenitors and conservation of the more primitive long-term bone marrow culture initiating cells (LTBMC- IC) was far superior in “stroma-noncontact” cultures supplemented with or without IL-3 than in “stroma-free” cultures supplemented with IL-3 alone or a combination of IL-3, LIF, G-CSF, and SCF. These studies indicate that human BM stroma produces soluble factors that can either alone or in synergy with defined cytokines (1) conserve primitive LTBMC- IC, (2) induce early differentiation of a fraction of the primitive progenitors, and (3) prevent their terminal differentiation. We show here that these stroma-derived factors are not likely to be the known early acting cytokines IL-3, SCF, LIF, or G-CSF. Characterization of the stroma-derived factor(s) may have important implications for clinically relevant studies, such as in vitro stem cell expansion in cancer treatment and gene therapy.

Developing a Model of Human Pluripotent to Hematopoietic Stem Cell Development in Mistrg Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4755-4755
Author(s):  
John Astle ◽  
Yangfei Xiang ◽  
Anthony Rongvaux ◽  
Carla Weibel ◽  
Henchey Elizabeth ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice

Abstract De novo generation of HSCs has been described as a "holy grail" of stem cell biology, however the factors required for converting human pluripotent stem cells (PSCs) to true hematopoietic stem cells (HSCs) capable of robust long-term engraftment have yet to be fully characterized. Two groups have shown that injection of PSCs into immunodeficient mice leads to teratomas containing niches producing hematopoietic progenitors capable of long-term engraftment. Once these hematopoietic progenitors and their microenvironments are better characterized, this system could be used as a model to help direct in vitro differentiation of PSCs to HSCs. Toward this end, we have injected human PSCs into immunodeficient mice expressing human rather than mouse M-CSF, IL-3, GM-CSF, and thrombopoietin, as well as both human and mouse versions of the "don't eat me signal" Sirpa (collectively termed MISTRG mice). These cytokines are known to support different aspects of hematopoiesis, and thrombopoietin in particular has been shown to support HSC maintenance, suggesting these mice may provide a better environment for human PSC-derived HSCs than the more traditional mice used for human HSC engraftment. The majority of teratomas developed so far in MISTRG contain human hematopoietic cells, and the CD34+ population isolated from over half of the teratomas contained hematopoietic colony forming cells by colony forming assay. These findings further corroborate this approach as a viable method for studying human PSC to HSC differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of stem cell factor on colony growth from acquired and constitutional (Fanconi) aplastic anemia

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 382-387 ◽  
Author(s):  
GP Bagnara ◽  
P Strippoli ◽  
L Bonsi ◽  
MF Brizzi ◽  
GC Avanzi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Aplastic Anemia ◽  
Stem Cell Factor ◽  
In Vitro Growth ◽  
Hematopoietic Progenitors ◽  
Gm Csf ◽  
Normal Controls ◽  
Marrow Cells

Abstract The aim of this study was to evaluate the effect of stem cell factor (SCF) on the in vitro growth of bone marrow hematopoietic progenitors from patients with acquired severe aplastic anemia (AA) or Fanconi's anemia (FA). For this purpose, we studied 11 patients with acquired AA (5 at diagnosis, 6 after ALG treatment), 12 patients with FA, and nine normal controls. Bone marrow cells were plated in vitro for colony- forming unit granulocyte-macrophage (CFU-GM) (in the presence of granulocyte-macrophage colony-stimulating factor [GM-CSF]), and for burst-forming unit-erythroid (BFU-E) and CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) colonies (in the presence of erythropoietin and interleukin-3 [IL-3]), with or without 20 ng/mL of SCF. In normal controls, SCF enhanced the growth of CFU-GM colonies from 103 to 263 (median), of BFU-E from 168 to 352, and of GEMM colonies from 6 to 38/10(5) cells plated. In patients with acquired AA, SCF induced a significant enhancement of BFU-E growth (8 to 29; P = .01) and allowed the formation of GEMM colonies that were not scored in baseline culture conditions (0 to 8; P = .01). CFU-GM growth was enhanced (4 to 20), but not significantly (P = .3). This was true both for patients at diagnosis and after antilymphocyte globulin treatment. By contrast, 10 of 12 FA patients grew no CFU-GM, BFU-E, or CFU-GEMM colonies, with or without SCF. In two FA patients (one transfusion- dependent and one transfusion-independent), an enhancement of CFU-GM and/or BFU-E was observed. The lack of response of hematopoietic progenitor cells from FA patients to GM-CSF+SCF or IL-3+SCF was not dependent on a defective expression of cytokine receptor messenger RNAs. Northern blot analysis showed in marrow cells from acquired AA and FA patients the presence of normal transcripts for alpha- and beta- chains of GM-CSF/IL-3 receptor and for c-kit protein. In conclusion, SCF promotes the in vitro growth of hematopoietic progenitors in patients with acquired AA, but not in patients with FA, pointing out the intrinsic nature of the defect in the latter disorder.

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