A Novel 3D Extracellular Matrix Model Enriching Human Acute Myeloid Leukemia Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4959-4959 ◽  
Author(s):  
Dandan Li ◽  
Tara L. Lin ◽  
Richard Hopkins ◽  
Omar S. Aljitawi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Leukemia Cell ◽  
Wharton’S Jelly ◽  
Leukemia Cells ◽  
Self Renewal ◽  
Wharton's Jelly ◽  
Cells Cultured

Abstract Background and objective: Acute myeloid leukemia (AML) develops from leukemia stem cells (LSCs), a small subset of leukemia cells possessing both self-renewal and multilineage differentiation potential similar to normal hematopoietic stem cells. The stem cell niche of the bone marrow microenvironment protects LSCs from chemotherapy, resulting in subsequent leukemia relapse. The study of AML LSCs in vitro is limited because of the lack of an ideal culture system mimicking the protective bone marrow microenvironment. The bone marrow stem cell niche is mainly composed of stromal cells, soluble cytokines and growth factors, as well as extracellular matrix (ECM). We therefore developed a 3-dimensional ECM model using decellularized Wharton's jelly from human umbilical cords to better characterize AML LSCs in vitro. Previously we have shown that leukemia cells grown in DWJM changed morphology to become spindle shaped and maintained viability but had decreased proliferation as measured by Alamar blue assay. Herein, we further characterize leukemia cells cultured in DWJM. Methods: Wharton's jelly decellularization process included multiple osmotic shock cycles using hypertonic and hypotonic solutions, a non-ionic detergent Triton-x, an anionic detergent sodium lauroyl succinate, and an enzyme digestion with recombinant endonuclease Benzonase™. We examined three human leukemia cell lines: HL60, Kasumi I and MV411. We characterized leukemia cell proliferation by CellTrace proliferation assay and phenotype by flow cytometry for stem cell markers. Serial colony forming unit (CFU) assays were used to test the self-renewal of leukemia cells. Results: CellTrace proliferation assay showed that, compared to cells in suspension, cells cultured in DWJM divided less frequently. To assess for LSCs properties, we measured the ALDH+ population by Aldefluor assay and found that the ALDH+ cells from Kasumi I and HL60 increased significantly in DWJM compared to suspension (p<0.05). Lastly, serial CFU showed significantly increased colony forming units in both primary and secondary plating in MV411 and HL60 cells cultured in DWJM versus suspension (P<0.05). CFU also increased in Kasumi-I cells cultured in DWJM versus suspension, though the increase was not statistically significant (P=0.08). Taken together, our findings suggest that leukemia cells cultured in DWJM demonstrated an increased self-renewal ability due to the enrichment of LSCs rather than progenitors. Conclusion: Decellularized Wharton's jelly matrix may serve as a practical in vitro ECM model to enrich for LSCs and study the ECM-LSC interactions. Disclosures No relevant conflicts of interest to declare.

scholarly journals Are MSCs Stem Cells? Demonstration of in Vitro and in Vivo Stem Cell Properties of Highly Enriched linneg/CD45neg/CD271pos/CD140alow/Neg Mesenchymal Stromal Cells from Adult Human Bone Marrow

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4374-4374
Author(s):  
Roshanak Ghazanfari ◽  
Hongzhe Li ◽  
Dimitra Zacharaki ◽  
Simón Méndez-Ferrer ◽  
Stefan Scheding
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Adult Human

Abstract Human bone marrow contains a rare population of non-hematopoietic mesenchymal stromal cells (BM-MSC) with multilineage differentiation capacity, which are essential constituents of the hematopoietic microenvironment. Self-renewal and differentiation are the two key properties of somatic stem cells, however, stem cell properties of human adult BM-MSC have not been demonstrated conclusively yet. We have previously shown that low/negative expression of PDGFRα on linneg/CD45neg/CD271pos cells identified a highly enriched population of primary BM-MSC in adult human bone marrow (Li et al. Blood, 2013, 122:3699). Based on this work, the current study aimed to investigate the in-vitro and in-vivo stem cell properties of this putative stromal stem cell population. The in-vitro clonogenic potential of freshly sorted human linneg/CD45neg/CD271pos/PDGFRlow/neg cells was evaluated by utilizing the CFU-F assay as well as the recently-developed mesensphere assay, which enables MSC amplification while preserving an immature phenotype (Isern et al, Cell Reports 2013, 30: 1714-24). Comparable colony frequencies were obtained with both assays (19.3 ± 2 and 17.5 ± 2.3 CFU-F and spheres per 100 plated cells, respectively, n=6, p=0.19). In order to test whether both assays identified the same population of clonogenic cells, colonies and spheres were replated under both conditions for up to three generations. The results showed comparable capacities of CFU-F and mesenspheres to form secondary and tertiary CFU-F and spheres. In-vitro self-renewal as indicated by increasing numbers of CFU-F and spheres (416.6 ± 431.7-fold and 49.5 ± 65.7-fold, respectively, n=3) was observed up to the third generation and decreased thereafter. The total number of generations was five (CFU-F) and six (spheres). In-vitro differentiation assays with both, CFU-F- and sphere-derived cells (tested until passage three) demonstrated tri-lineage differentiation potential (adipocytes, osteoblasts, chondrocytes). In addition, CFU-Fs and spheres had comparable surface marker profiles (CD73, CD90, CD105, and HLA-ABC positive; CD31, CD34 and HLA-DR negative), except for CD90, which was higher expressed on CFU-Fs. To investigate in-vivo self-renewal and differentiation potential of the putative stromal stem cells, linneg/CD45neg/CD271pos/PDGFRlow/neg -derived CFU-F and spheres were serially transplanted s.c into NSG mice. After 8 weeks, implants were harvested, human cells were FACS-isolated (CD90 and CD105 expression), and re-assayed under CFU-F and sphere conditions. Whereas in-vivo self-renewal of CFU-F could not be shown (111.5 ± 36 –fold decrease in total CFU-F numbers after primary transplantation, n=3), sphere self-renewal was clearly demonstrated by increased numbers of spheres after primary as well as secondary transplantation (1.13 ± 0.05 and 2.06 ± 0.26 –fold, respectively, n=3), which is remarkable given the fact that the number of recovered human cells is underestimated due to the isolation approach. Here, confirming GFP-marking experiments are ongoing. Finally, preliminary data indicate that linneg/CD45neg/CD271pos/PDGFRlow/neg –derived spheres display full in-vivo differentiation capacity in primary and secondary transplantations. Taken together, our data demonstrate - for the first time - that primary human linneg/CD45neg/CD271pos/PDGFRlow/neg cells meet stringent stem cell criteria, i.e. in-vitro and in-vivo self-renewal and differentiation. These findings answer the long-open question of the potential stem cell properties of adult human MSC and will enable to better understand the properties of native BM-MSC and their biological role in the bone marrow. Disclosures No relevant conflicts of interest to declare.

scholarly journals Intrinsic Cellular Responses of Human Wharton’s Jelly Mesenchymal Stem Cells Influenced by O2-Plasma-Modified and Unmodified Surface of Alkaline-Hydrolyzed 2D and 3D PCL Scaffolds

2019 ◽  
Vol 10 (4) ◽  
pp. 52 ◽  
Author(s):  
Inthanon ◽  
Janvikul ◽  
Ongchai ◽  
Chomdej
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Attachment ◽  
Physicochemical Characterization ◽  
Wharton’S Jelly ◽  
Wharton's Jelly ◽  
Differentiation Inducers ◽  
2D And 3D

Polycaprolactone (PCL), a hydrophobic-degradable polyester, has been widely investigated and extensively developed, to increase the biocompatibility for tissue engineering. This research was the first trial to evaluate the intrinsic biological responses of human Wharton’s Jelly Mesenchymal Stem Cells (hWJMSCs) cultured on alkaline hydrolysis and low-pressure oxygen plasma modified 2D and 3D PCL scaffolds, without adding any differentiation inducers; this has not been reported before. Four types of the substrate were newly established: 2D plasma-treated PCL (2D-TP), 2D non-plasma-treated PCL (2D-NP), 3D plasma-treated PCL (3D-TP), and 3D non-plasma-treated PCL (3D-NP). Physicochemical characterization revealed that only plasma-treated PCL scaffolds significantly increased the hydrophilicity and % oxygen/carbon ratio on the surfaces. The RMS roughness of 3D was higher than 2D conformation, whilst the plasma-treated surfaces were rougher than the non-plasma treated ones. The cytocompatibility test demonstrated that the 2D PCLs enhanced the initial cell attachment in comparison to the 3Ds, indicated by a higher expression of focal adhesion kinase. Meanwhile, the 3Ds promoted cell proliferation and migration as evidence of higher cyclin-A expression and filopodial protrusion, respectively. The 3Ds potentially protected the cell from apoptosis/necrosis but also altered the pluripotency/differentiation-related gene expression. In summary, the different configuration and surface properties of PCL scaffolds displayed the significant potential and effectiveness for facilitating stem cell growth and differentiation in vitro. The cell–substrate interactions on modified surface PCL may provide some information which could be further applied in substrate architecture for stem cell accommodation in cell delivery system for tissue repair.

Stromal Cell-Derived Secreted Factors Contribute to the Innate Imatinib Resistance of Leukemia Stem Cells In a Genetically Defined Murine Model of Chronic Myeloid Leukemia Blast Crisis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 204-204
Author(s):  
C. Ronald Geyer ◽  
Michael Szeto ◽  
Ashton Craven ◽  
Marciano D. Reis ◽  
David P. Sheridan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cell ◽  
Blast Crisis ◽  
Conditioned Media ◽  
Imatinib Resistance ◽  
Self Renewal ◽  
Soluble Factors

Abstract Abstract 204 Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease. In vitro studies of human CML cell lines and CD34+ cells isolated from CML patients, have shown that bone marrow stromal cell factor (BMSF) conditioned media can maintain important pro-survival and self-renewal activities downstream of BCR-ABL in the presence of TKIs, suggesting a role for secreted BMSFs in innate resistance to BCR-ABL kinase inhibition. However, the ability of BMSFs to maintain the leukemic potential of CML stem cells upon exposure to TKIs has not been reported. We used a standard murine retroviral transduction system to model CML blast crisis (BC-CML) and obtain cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 72 hrs in BMSF conditioned media across a range of concentrations (0% - 50%) in the presence and absence of imatinib (0 - 1000 nM). BMSF conditioned media reduced the cytotoxic effects of imatinib on GFP+/YFP+ LSKs as assessed by cell counts, trypan blue viability assays, and Annexin V expression by flow cytometry. Furthermore, BMSF conditioned media reduced the inhibitory effects of imatinib on GFP+/YFP+ LSK colony formation in methylcellulose, and beta-catenin expression as assessed by flow cytometry. These observations strongly suggest that signaling by stromal cell-derived soluble factors protects BC-CML stem cells from imatinib therapy by re-activating pro-survival and self-renewal pathways. The ability of BMSFs to reduce the inhibitory effect of imatinib on BC-CML stem cell self-renewal in vivo was assessed by performing secondary transplantation assays. GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to BMSF conditioned media in the presence and absence of 1000 nM imatinib. Survival after transplantation was compared in cohorts of 5 mice per experimental condition: Group 1 (0% BMSF, 0 nM imatinib), Group 2 (50% BMSF, 0 nM imatinib), Group 3 (50% BMSF, 1000 nM imatinib) and Group 4 (0% BMSF, 1000 nM imatinib). Survival was significantly prolonged in Group 4 mice treated with 1000 nM imatinib and this effect was abrogated by treatment with 50% BMSF conditioned media, indicating that cell-derived soluble factors contribute to maintaining BC-CML stem cell function in the presence of imatinib. Our findings strongly suggest that signaling by soluble BMSFs plays an important role in the innate imatinib resistance of CML stem cells, implicating these factors in disease relapse. Genetically defined murine models of CML provide a powerful in vivo system to identify and target soluble factors that contribute to stromal-mediated cytoprotection of CML stem cells from TKIs. Disclosures: No relevant conflicts of interest to declare.

ATRA and the RARα Specific Agonist, NRX195183, Have Opposing Effects on the In Vitro Clonogenicity of Pre-Leukemic Murine AML1-ETO Bone Marrow Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 999-999
Author(s):  
Lynette C.Y. Chee ◽  
Jean Hendy ◽  
Louise Purton ◽  
Grant A. McArthur
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Sustained Remission ◽  
Self Renewal ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Abstract 999 All-trans retinoic acid (ATRA) is used successfully to treat acute promyelocytic leukemia (APML), however, to date it has not shown promise in treating other AML subtypes. ATRA has been shown to enhance hematopoietic stem cell (HSC) self-renewal (requiring RARγ activation) but promotes differentiation of myeloid progenitors likely through RARα activation. We hypothesized that (1) the lack of success of ATRA in treating other AML subtypes may be due to the potential ability of ATRA to enhance self-renewal of the leukemic stem cell and (2) the use of a specific RARα agonist may have more promise in enhancing AML differentiation. We therefore compared the effects of pharmacological levels (1μM) of ATRA and an RARα-specific agonist, NRX195183, on bone marrow cells harvested from a Cre-inducible conditional AML1-ETO (AE) knock-in murine model. AE cells cultured for 2 weeks with ATRA showed significant reductions in the proportions of mature myeloid cells (Gr1brightCD11b+) by fluorescence activated cell sorting (FACS) (DMSO: 14.2±4.3%, ATRA: 4.0±1.6%, p=0.04, n=4). By 4 weeks of culture, ATRA-treated AE cells had increased blast and reduced maturing myeloid cell proportions (Blasts %: DMSO 70.2 ± 3.0, ATRA 95.3 ± 1.2, p=0.08; Intermediate %: DMSO 14.3 ± 2.6, ATRA 3.8 ± 1.0, p=0.01; Neutrophils %: DMSO 2.3± 1.0, ATRA 0.3 ± 0.2, p=0.07, n=6). Furthermore, ATRA potentiated the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells: DMSO 505.8±337.0, ATRA 4394±388.9, p=0.001; n=6). In contrast, AE cells cultured for 2 weeks with NRX195183 showed significant increases in the proportions of mature myeloid cells by FACS (DMSO: 15.8±3.5%, NRX195183 26.7±3.0%, p=0.03; n=5). By 4 weeks of culture, NRX195183-treated AE cells had decreased blast and increased maturing myeloid cell proportions (Blasts %: DMSO 82.4±3.0, NRX195183 58.8±9.1, p=0.03; Intermediate %: DMSO 14.5±2.5, NRX195183 29.0±6.8, p=0.07; Neutrophils %: DMSO 1.6±0.8, NRX195183 8.2±4.7 p=ns; DMSO n=8, NRX195183 n=5). Moreover, NRX195183 reduced the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells DMSO 554.8±252.6, NRX195183 82.6±61.6, p=0.05; n=8). Short-term in vivo transplants of fetal liver cells overexpressing the truncated AE gene, AE9a, into sublethally irradiated recipients revealed similar findings in the NRX195183-treated mice with a decrease in blasts and an increase in mature neutrophils in the peripheral blood on morphological analysis after 4 weeks of treatment (Blasts x106/ml: DMSO 3.1±1.0, NRX195183 0.9±0.3, p=0.08; Neutrophils x106/ml: DMSO 0.5±0.1, NRX195183 0.8±0.1, p=0.04; DMSO n=16, NRX195183 n=11). Taken together, these findings support a model whereby ATRA promotes self-renewal of leukemic blasts whilst NRX195183 has the opposing effect. To understand the mechanism by which ATRA promotes self-renewal in AE cells, we performed genome-wide gene expression analyses on the ATRA- versus control-treated AE cells. This revealed 16 differentially upregulated genes after 24 hours of treatment. Using Ingenuity Pathway Analysis, the top scoring network in the ATRA-treated AE cells was cell-to-cell signalling and interaction (p=1.1E-7-2.4E-3), lipid metabolism (p=2.3E-7-2.0E-3) and small molecule biochemistry (p=2.3E-7-2.1E-3); SERPINE1 and BMP2 were the genes with the highest connectivity within the network interacting with molecules known for their roles in tumorigenesis, including AKT, NF-kβ complex and TGFβ1. SERPINE1 upregulation has been shown to be RARα-mediated whilst BMP2 has been shown to be a RARγ-regulated gene. Interestingly, the specific RARγ agonist, NRX204723, had no effect on the clonogenic potential of these AE progenitors thus raising the hypothesis that both RARα and RARγ activation are required to promote self-renewal of the AE progenitors. Further studies using both RARα/RARγ agonists are warranted to assess if the ATRA effects on AE cells are phenocopied. Collectively, these findings reveal the contrasting roles of specific RARα activation in promoting loss of self-renewal ability and enhancing differentiation in the AE cells whilst ATRA promotes clonogenicity of these cells. This has potential significant implications in AML treatment as specific RARα agonists may be beneficial in improving the efficacy of current treatment modalities to achieve sustained remission in other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1688-1688 ◽  
Author(s):  
Noriko Miyake ◽  
Ann C.M. Brun ◽  
Mattias Magnusson ◽  
David T. Scadden ◽  
Stefan Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

Resistance To Chemotherapy In Leukemia Cells Grown On An Extracellular Matrix-Based Leukemia Model Derived From Wharton’s Jelly

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1388-1388 ◽  
Author(s):  
Dandan Li ◽  
Tara L. Lin ◽  
Da Zhang ◽  
Linheng Li ◽  
Richard A. Hopkins ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Extracellular Matrix ◽  
Cell Lines ◽  
Adhesion Molecule ◽  
Neutralizing Antibody ◽  
Wharton’S Jelly ◽  
Leukemia Cells ◽  
Wharton's Jelly ◽  
Cells Cultured

Abstract Background A growing body of evidence implicates chemotherapy-resistant and quiescent leukemia stem cells (LSC) in the relapse of Acute Myeloid Leukemia (AML). Similar to hematopoietic stem cells, these LSCs reside in the bone marrow microenvironment in niches comprised of other cells or extracellular matrix (ECM). There are various components of the LSC niche that ensures LSC quiescence through expression of cell adhesion molecules. Many in vitro models of AML do not incorporate features of the bone marrow microenvironment or LSC niche, and thus, are less representative of the in vivo leukemic state. In our published work and that of others, AML cells co-cultured in a stromal-based model demonstrated increased resistance to chemotherapy as compared to conventional suspension cultures. In our prior experience, we decellularized Wharton’s jelly, the gelatinous material in the umbilical cord, to obtain a matrix material devoid of cells. This Wharton’s jelly matrix is composed of collagen, fibronectin, and hyaluronic acid with its CD44 receptor, which are components of bone marrow extracellular matrix. Therefore, we hypothesized that decellularized Wharton’s jelly matrix (DWJM) could serve as an ECM-based model to study acute myeloid leukemia behavior and chemotherapy sensitivity. We hypothesized that leukemia cells cultured in DWJM will be more resistant to chemotherapy compared to leukemic cells grown in suspension. Materials and methods We used DWJM as a scaffolding material to culture leukemia cells from AML cell lines (Kasumi-1, HL-60, and MV411). We evaluated the effect of DWJM on leukemia cell growth including viability by trypan blue exclusion, proliferation by alamarBlue, and differentiation by flow cytometry. We also examined the expression pattern of the adhesion molecule CD44 by Western blot and immunofluorescence while leukemia cells are growing in DWJM. Cell cycle was analyzed by flow cytometry following cell staining with propidium iodide. Furthermore, we compared the different apoptotic responses, by flow cytometry, of leukemia cells cultured in suspension or DWJM to doxorubicin treatment as well as to the combination of doxorubicin and CD44 neutralizing antibody. Results Leukemic cell lines demonstrated successive decrease in their proliferation over time. This was associated with decreased viability, albeit to a lower extent. Cell cycle analysis showed that 86% of MV411 cells and 70% of Kasumi-1 cells cultured on DWJM were arrested in G0/G1 state consistent with quiescence. Additionally, no significant differentiation in cell lines occurred during culture in DWJM. In Kasumi I cells, the adhesion molecule CD44 was highly immunoexpressed when cultured in DWJM, while no CD44 was expressed by cell in suspension as measured by Western blot. Interestingly, leukemia cells seeded on DWJM changed their morphology, forming spindle-shaped cells associated with the expected round cell morphology. In terms of response to doxorubicin treatment measured by apoptosis flow cytometry, MV411 cells cultured in DWJM were significantly more resistant to doxorubicin compared to cells in suspension, while Kasumi I cells were significantly more sensitive to doxorubicin when grown in DWJM. Interestingly, when doxorubicin was combined with a CD44 neutralizing antibody given in similar doses, MV411 demonstrated significant increase in cell death while Kasumi I demonstrated significant decrease in cell death. Conclusion Leukemia cell lines can be cultured in DWJM without undergoing differentiation. Leukemic cell growth in this ECM model was characterized by decreased proliferation, dormancy, changes in morphology by acquiring a spindle-shaped appearance, increased expression of the cell adhesion molecule CD44, and by acquiring drug-resistance characteristics in MV411 cell line. CD44 neutralizing antibody did overcome chemotherapy resistance in MV411 but not in Kasumi-1. This data suggests an important role for CD44 in mediating chemotherapy sensitivity in an ECM-based model of AML. Disclosures: No relevant conflicts of interest to declare.

Coordinated Regulation of Hematopoietic and Mesenchymal Stem Cells in a Bone Marrow Niche.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2-2 ◽  
Author(s):  
Simón Méndez-Ferrer ◽  
Tatyana V. Michurina ◽  
Francesca Ferraro ◽  
Amin Mazloom ◽  
Ben MacArthur ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Osteoblastic Differentiation ◽  
Cell Depletion ◽  
Self Renewal

Abstract Abstract 2 Despite their therapeutic potential, mesenchymal stem cells (MSCs) remain poorly defined owing to their heterogeneity, the inability to assess in vivo self-renewal and the scarcity of markers allowing their identification, isolation and genetic manipulation. In the bone marrow (BM) of Nestin (Nes)-Gfp transgenic mice, CD31− CD45− GFP+ peri-vascular cells expressing endogenous nestin are associated with hematopoietic stem cells (HSCs) and innervated by fibers from the sympathetic nervous system (SNS). Flow cytometry sorting of BM CD45− Nes:GFP+ and CD45− Nes:GFP− cells has revealed that Nes:GFP+ cells, despite their rarity (4.0 ± 0.6% CD45− cells), contain all the colony-forming unit-fibroblastic (CFU-F) activity and have the exclusive capacity of forming self-renewing, multipotent clonal spheres that differentiate robustly along osteoblastic, chondrocytic and adipocytic lineages. To test in vivo self-renewal, single spheres derived from Nes-Gfp / Col2.3-Cre / R26R triple-transgenic animals were allowed to attach to phosphocalcic ceramic ossicles that were subcutaneously implanted into littermate mice that did not carry the transgenes. Histological analyses after 2 months revealed the presence of β-galactosidase+ osteoblasts (OBs) derived from Nes:GFP+ cells and not from 30,000 control CD45− Nes:GFP− cells. Hematopoietic areas were associated with Nes:GFP+ cells, that yielded per ossicle 310 ± 32 GFP+ secondary spheres (n=6), 38.6 ± 1.9% of which showed spontaneous multilineage differentiation into Col2.3+ OBs and Oil Red O+ adipocytes. Single secondary spheres subjected to a subsequent round of transplantation yielded after 8 months 8,557 ± 537 GFP+ spheres per ossicle (n = 7), which also generated Col2.3+ OBs, as a further proof of their self-renewal, osteoblastic differentiation potential and donor origin. Lineage-tracing studies in Nes-Cre / R26R mice have revealed the contribution of nestin-expressing cells in endochondral and membranous ossification. Administration of tamoxifen to adult Nes-CreERT2 mice bred to different reporter lines revealed that adult nestin-expressing BM cells could generate OBs, chondrocytes and osteocytes after 8-month chasing, suggesting an active role for adult nestin+ MSCs in physiological bone turnover. Genome-wide comparison analyses have shown that BM CD45− Nes:GFP+ cells are distinct from other stem cells but closest to in vitro expanded MSCs. Applying gene ontology analyses, metabolic and cell cycle genes were up- and down-regulated, respectively, in BM CD45− Nes:GFP+ cells. We have studied gene regulation, cell cycle and fate in response to granulocyte-colony stimulating factor (G-CSF), parathormone (PTH) and signals from the SNS, stimuli that regulate both hematopoietic and mesenchymal lineages in the BM. Cell cycle studies from FACS-sorted, flushed BM samples have confirmed that CD45− Nes:GFP+ cells are much more quiescent (90% G0/G1) than CD45− Nes:GFP− cells (58% G0/G1) but are selectively induced to proliferate after chemical sympathectomy (61% G0/G1) or PTH (70% G0/G1) administration in mice (n = 4–5). The inhibitory effects of the SNS and G-CSF (95% G0/G1) on BM CD45− Nes:GFP+ cells were not limited to cell cycle but also involved osteoblastic differentiation and expression of HSC maintenance genes. By contrast, in vivo or in vitro treatment with PTH selectively induced proliferation and osteoblastic differentiation of CD45− Nes:GFP+ cells, which express PTH receptor 1. We generated selective cell depletion models by intercrossing Nes-Cre and Nes-CreERT2 mice with a Cre-inducible diphtheria toxin receptor line (iDTR). In both models, HSC numbers decreased by ∼ 50% in the BM and increased in the spleen, an effect directly caused by selective BM cell depletion, as per in vitro experiments. In the more specific Nes-CreERT2 model, this effect was specific for HSCs and not for more mature progenitors. Cell depletion in Nes-Cre / iDTR and Nes-CreERT2 / iDTR mice reduced homing of hematopoietic progenitors by 73 and 90%, respectively. Finally, combined two-photon and confocal microscopy of the calvarial BM has demonstrated that highly purified, labeled HSCs rapidly (≤ 2h) home near Nes:GFP+ cells. Thus, cytokines, hormones, and the SNS regulate both HSC maintenance and bone formation in the BM stem cell niche through direct control of nestin-expressing MSCs. These results uncover an unprecedented partnership between two distinct somatic stem cell types and argue for a unique peri-vascular niche in the BM formed by MSC-HSC pairs. Disclosures: Scadden: Fate Therapeutics: Consultancy. Frenette:Glycomimetic: Research Funding.

scholarly journals Stem cell factor increases colony-forming unit-spleen number in vitro in synergy with interleukin-6, and in vivo in Sl/Sld mice as a single factor

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 913-919 ◽  
Author(s):  
DM Bodine ◽  
D Orlic ◽  
NC Birkett ◽  
NE Seidel ◽  
KM Zsebo
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Growth Factors ◽  
Stem Cell Factor ◽  
Bone Marrow Cells ◽  
Cells Cultured

Abstract Hematopoiesis is thought to be modulated by interactions of progenitor cells with hematopoietic growth factors. We have shown that colony- forming units-spleen (CFU-S) and repopulating stem cells require interleukin-3 (IL-3) to survive in vitro, and that CFU-S number and long-term repopulating ability can be increased by culture in the combination of IL-3 and IL-6. In this report, we describe the effects of stem cell factor (SCF) on CFU-S and repopulating stem cells. Injection of SCF into anemic Sl/Sld mice caused a twofold and 20-fold increase in CFU-S number in the bone marrow and spleen of treated animals, respectively. After 6 days in suspension culture, CFU-S number increased threefold in cultures supplemented with SCF and IL-6, or SCF, IL-3, and IL-6 relative to the number at day 0. The long-term repopulating ability of cells cultured in SCF, IL-3, and IL-6 was approximately sevenfold better than that of cells cultured in IL-3 or SCF. Similar experiments were performed on populations of bone marrow cells enriched for, or depleted of, CFU-S by elutriation and lineage subtraction. The combination of SCF and IL-6 increased CFU-S number approximately fourfold to eightfold in the CFU-S-enriched fraction, but had no effect on the CFU-S-depleted cells. These results show that SCF alone can increase CFU-S number in vivo, and in combination with other growth factors increases CFU-S numbers in vitro.

scholarly journals Stem cell factor increases colony-forming unit-spleen number in vitro in synergy with interleukin-6, and in vivo in Sl/Sld mice as a single factor

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 913-919 ◽  
Author(s):  
DM Bodine ◽  
D Orlic ◽  
NC Birkett ◽  
NE Seidel ◽  
KM Zsebo
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Growth Factors ◽  
Stem Cell Factor ◽  
Bone Marrow Cells ◽  
Cells Cultured

Hematopoiesis is thought to be modulated by interactions of progenitor cells with hematopoietic growth factors. We have shown that colony- forming units-spleen (CFU-S) and repopulating stem cells require interleukin-3 (IL-3) to survive in vitro, and that CFU-S number and long-term repopulating ability can be increased by culture in the combination of IL-3 and IL-6. In this report, we describe the effects of stem cell factor (SCF) on CFU-S and repopulating stem cells. Injection of SCF into anemic Sl/Sld mice caused a twofold and 20-fold increase in CFU-S number in the bone marrow and spleen of treated animals, respectively. After 6 days in suspension culture, CFU-S number increased threefold in cultures supplemented with SCF and IL-6, or SCF, IL-3, and IL-6 relative to the number at day 0. The long-term repopulating ability of cells cultured in SCF, IL-3, and IL-6 was approximately sevenfold better than that of cells cultured in IL-3 or SCF. Similar experiments were performed on populations of bone marrow cells enriched for, or depleted of, CFU-S by elutriation and lineage subtraction. The combination of SCF and IL-6 increased CFU-S number approximately fourfold to eightfold in the CFU-S-enriched fraction, but had no effect on the CFU-S-depleted cells. These results show that SCF alone can increase CFU-S number in vivo, and in combination with other growth factors increases CFU-S numbers in vitro.

scholarly journals Cell Source-Dependent In Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus dromedarius

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1918
Author(s):  
Young-Bum Son ◽  
Yeon Ik Jeong ◽  
Yeon Woo Jeong ◽  
Mohammad Shamim Hossein ◽  
Per Olof Olsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Synovial Fluid ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Chondrocyte Differentiation ◽  
Differentiation Potential ◽  
Proliferation Capacity

Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular characteristics and chondrocyte differentiation potential were not reported in either of the camel stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast-like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM-MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF-MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes in camels.

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