stemspan serum free expansion media ii (sfem ii)  (STEMCELL Technologies Inc)

Journal: STAR Protocols

Article Title: FACS and immunomagnetic isolation of early erythroid progenitor cells from mouse fetal liver

doi: 10.1016/j.xpro.2021.101070

Figure Lengend Snippet:

Article Snippet: Stemspan Serum-free expansion media (SFEM) II , Stem Cell Technologies , Cat#:09655.

Techniques: Recombinant, Saline, Magnetic Beads, Selection, Gene Expression, Cell Culture, Transferring, Membrane

Journal: STAR Protocols

Article Title: FACS and immunomagnetic isolation of early erythroid progenitor cells from mouse fetal liver

doi: 10.1016/j.xpro.2021.101070

Figure Lengend Snippet:

Article Snippet: Stemspan Serum-free expansion media (SFEM) II , Stem Cell Technologies , Cat#:09655.

Techniques: Concentration Assay, Recombinant

Journal: Stem Cells Translational Medicine

Article Title: Ex Vivo Expansion of CD34 + CD90 + CD49f + Hematopoietic Stem and Progenitor Cells from Non‐Enriched Umbilical Cord Blood with Azole Compounds

doi: 10.1002/sctm.17-0251

Figure Lengend Snippet: Identification of a novel small molecule, C7 that is a structural analog of the p38‐MAPK inhibitor SB203580, which could expand HPC from non‐enriched UCB–MNC. (A): Fold expansion of viable (7AAD – ) CD45 + CD34 + CD38 – CD45RA – HPC and TNC in cultures that lasted for 11 days with animal component free media (ACF) media, cytokine, and respective small molecule being replenished at day 7. SB203580, dimethyl sulfoxide, and cytokines alone in ACF media served as the reference compound, vehicle and blank control, respectively. The dashed black and blue line represents >2.0‐fold expansion of HPC and TNC, respectively compared to cytokine control. Expansion data of HPC and TNC for lead compound C7 is highlighted with red circles. *, p < .001 compared to all other conditions for HPC expansion. *, p < .05 compared to all other conditions for TNC expansion. Data represents mean ± SD for n = 3. (B): Representative flow cytometer contour plots depicting CD34 + CD38 – population which is a subset of the viable (7AAD – ) CD45 + cells of (i) thawed non‐selected UCB MNC at day 0 followed by culturing for 11 days in (ii) cytokine control and (iii) 5.0 µM of C7 supplemented with cytokines using serum free expansion media. Media, cytokines, and C7 were replenished at day 7. (C): Graphical summary of the structural activity relationship of the azole analogs investigated in inducing the ex vivo expansion of HPC from UCB–MNC. (D): Chemical structures, IUPAC name and molecular weight of SB203580 (parent compound) and C7 (top lead compound). Abbreviations: HPC, hematopoietic progenitor cells; MNC, mononucleated cells; TNC, total nucleated cell; UCB, umbilical cord blood.

Article Snippet: UCB–MNC were cultured at an empirically determined optimal density of 4.0 × 10 5 cells/ml without any cell surface marker dependent stem cell enrichment in StemSpan‐Serum Free Expansion Media (SFEM) or StemSpan‐Animal Component Free Media (ACF) (STEMCELL Technologies, Canada) supplemented with human cytokine cocktail of 100 ng/ml stem cell factor (SCF) (PeproTech, USA) and thrombopoietin (TPO) (PeproTech); 50 ng/ml FLT‐3 Ligand (FLT‐3L) (PeproTech); and 20 ng/ml insulin‐like growth factor binding protein‐2 (IGFBP‐2) (R&D Systems, USA).

Techniques: Control, Flow Cytometry, Activity Assay, Ex Vivo, Molecular Weight

Journal: Stem Cells Translational Medicine

Article Title: Ex Vivo Expansion of CD34 + CD90 + CD49f + Hematopoietic Stem and Progenitor Cells from Non‐Enriched Umbilical Cord Blood with Azole Compounds

doi: 10.1002/sctm.17-0251

Figure Lengend Snippet: Expansion of HPC in presence of C7 with varying cytokine combination and in comparison to established expansion technologies. (A): Fold expansion of viable (7AAD – ) CD45 + CD34 + CD38 – CD45RA – HPC in cultures that lasted for 11 days with ACF media, different combinations of cytokines with and without 5.0 µM of C7. Media, group specific cytokine combinations and C7 were replenished at day 7. The concentrations of each cytokine are as follows: S represents SCF at 100 ng/ml; T represents TPO at 100 ng/ml; F represents FLT‐3L at 50 ng/ml; and IG represents IGFBP‐2 at 20 ng/ml. *, p < .001 compared to all other conditions. **, p < .05 when compared between indicated groups. Data represents mean ± SD for n = 3. (B): Ex vivo expansion of viable (7AAD – ) CD45 + CD34 + CD38 – CD45RA – HPC, lymphoid (CLP: CD45 + CD34 + CD38 – CD7 + ) and myeloid progenitors (CMP: CD45 + CD34 + CD13 + CD33 + ) from two separate UCB units were cultured with 5.0 µM of C7, cytokines and MSC. The expansion cultures lasted for 11 days with SFEM media, cytokine, and C7 replenishment being done on day 7. Cytokines alone in media served as the blank control. *, p < .0001 compared to respective population of all groups. *, p < .05 compared to respective population of all groups. Data represent mean ± SEM for n = 6. (C): Expansion of CD34 + bright (early HPC: CD45 + CD34(bright) + CD38 – CD45RA – ) and CD34 + dim (early HPC: CD45 + CD34(dim) + CD38 – CD45RA – ) progenitors when cultured in ACF media containing 5.0 µM of C7, or commercially available small molecules in presence of basal cytokines. The expansion cultures lasted for 11 days with ACF media, cytokine, and small molecules replenishment being done on day 7. Cytokines alone in media served as the blank control. The concentrations of each small molecule are as follows: S represents SR‐1 at 1.0 µM; U represents UM171 at 50.0 nM; and N represents Nicotinamide at 5.0 mM. *, p < .01 compared to respective population in all groups. Data represents mean ± SD for n = 3. (D): Ex vivo expansion of viable (7AAD – ) CD45 + CD34 + CD38 – CD45RA – HPC when cultured in presence of 5.0 µM of each of the stated kinase modulators and basal cytokine cocktail. The expansion cultures lasted for 11 days with media, cytokine, and respective small molecule being replenished once on day 7. The control was SFEM media containing cytokines alone. The reference compound, SB203580, and vehicle control, DMSO, was also included. *, p < .01 compared to all other compounds and controls. Data represents mean ± SD for n = 3. (E): Expansion of viable CD45 + CD34 + CD38 – CD45RA – HPCs when cultures were initiated from magnetically purified CD34 + grafts. The expansion cultures lasted for 11 days with ACF media, cytokine, and 5.0 µM of C7 replenishment being done on day 7. *, p < .0001 compared between the groups C7 and cytokines and cytokine control. Data represents mean ± SEM for n = 6. (F): Experiment 1: Ex vivo expansion of CFU–GM when two separate UCB units without pre‐selection of stem cells were cultured in 5.0 μM of C7 and basal cytokines. The expansion cultures lasted for 10–11 days with SFEM, cytokine, and C7 replenishment being done on day 7. SB203580, DMSO, and cytokines alone in SFEM served as the reference compound, vehicle and blank control, respectively. *, p < .01 compared to SB203580, DMSO, and Cytokine control. Data represent mean ± SEM for n = 6. Experiment 2: Fold expansion of CFU–GM in cultures that lasted for 7 and 11 days. ACF media, cytokine, and 5.0 μM of C7 were replenished on day 7 for cultures lasting till day 11. Cytokines alone in ACF media served as the blank control. p values generated from Student's t test among indicated experimental groups are shown in the graph for the stated n values. Data represents mean ± SD for n = 3. Experiment 3: Ex vivo expansion of CFU–GM when UCB–MNC were cultured in SFEM or ACF media containing 5.0 μM of C7 in presence of basal cytokines. The expansion cultures lasted for 10–11 days with SFEM/ACF media, cytokine, and C7 replenishment being done on day 7. Cytokines alone in SFEM/ACF media served as the blank control. p values generated from Student's t test among indicated experimental groups are shown in the graph for the stated n values. Data represents mean ± SD for n = 3. (G): Ex vivo expansion of CFU–GEMM and BFU‐E when UCB–MNC were cultured in SFEM media containing 5.0 μM of C7 in presence of basal cytokines. The expansion cultures lasted for 11 days with SFEM, cytokine, and C7 replenishment being done on day 7. Cytokines alone in SFEM served as the blank control. *, p < .0001 and **, p < .05 compared to cytokine control for CFU–GEMM and BFU‐E expansion, respectively. Data represents mean ± SEM for n = 6. Abbreviations: ACF, animal component free media; BFU‐E, erythroid burst‐forming units; CFU, colony forming unit; DMSO, dimethyl sulfoxide; FLT‐3L, FLT‐3 Ligand; GEMM, granulocyte‐erythrocyte‐monocyte‐megakaryocyte; GM, granulocyte, monocyte; HPC, hematopoietic progenitor cells; IGFBP‐2, insulin‐like growth factor binding protein‐2; MNC, mononucleated cells; MSC, mesenchymal stromal cell; SCF, stem cell factor; SFEM, serum free expansion media; TPO, thrombopoietin; UCB, umbilical cord blood.

Article Snippet: UCB–MNC were cultured at an empirically determined optimal density of 4.0 × 10 5 cells/ml without any cell surface marker dependent stem cell enrichment in StemSpan‐Serum Free Expansion Media (SFEM) or StemSpan‐Animal Component Free Media (ACF) (STEMCELL Technologies, Canada) supplemented with human cytokine cocktail of 100 ng/ml stem cell factor (SCF) (PeproTech, USA) and thrombopoietin (TPO) (PeproTech); 50 ng/ml FLT‐3 Ligand (FLT‐3L) (PeproTech); and 20 ng/ml insulin‐like growth factor binding protein‐2 (IGFBP‐2) (R&D Systems, USA).

Techniques: Comparison, Ex Vivo, Cell Culture, Control, Purification, Selection, Generated, Binding Assay

Journal: Stem Cells Translational Medicine

Article Title: Ex Vivo Expansion of CD34 + CD90 + CD49f + Hematopoietic Stem and Progenitor Cells from Non‐Enriched Umbilical Cord Blood with Azole Compounds

doi: 10.1002/sctm.17-0251

Figure Lengend Snippet: C7 augmented expansion of rare CD34 + CD90 + CD49 + hematopoietic stem and progenitor cells when cultures were initiated with non‐enriched umbilical cord blood (UCB)–mononucleated cells (MNC). (A): Representative flow cytometer dot plots depicting (a) CD90 + CD49f – (region depicted with *); (b) CD90 + CD49f + (region depicted with **) and (c) CD90 – CD49f + (region depicted with ***) population which are subsets of CD45 + CD34 + CD38 – CD45RA – cells of (i) thawed UCB MNC at 0 hours followed by culturing for 10 days in (ii) cytokine control and (iii) 5.0 µM of C7 supplemented with cytokines using serum free expansion media (SFEM). Media, cytokines, and C7 were replenished at day 7. (B): Expansion of viable (7AAD – ) HSC1 and HSC2 with phenotypic expression of CD45 + CD34 + CD38 – CD45RA – CD90 + CD49f – and CD45 + CD34 + CD38 – CD45RA – CD90 + CD49f + respectively when cultures were initiated with non‐selected UCB–MNC. The expansion cultures lasted for 10 days, with SFEM, cytokine, and 5.0 µM of C7 replenishment being done on day 7. Cytokines alone in media served as the blank control. *, p < .001 compared to respective population in each group. Data represents mean ± SD for n = 3. (C): Expansion of mature myeloid and lymphoid lineage cells in C7 and cytokine control cultures over 11 days. These MNC expansion cultures were supplemented with animal component free media, cytokine, and 5.0 µM of C7 at day 7. Myeloid lineage consisted of CD45 + CD33 + monocytes, CD45 + CD13 + CD15 + granulocytes, and CD45 + CD41a + CD61 + megakaryocytes. Lymphoid lineage consisted of CD45 + CD3 + T cells, CD45 + CD19 + B cells, and CD45 + CD56 + NK cells. *, p < .001 compared to respective population in each treatment group. Data represents mean ± SD for n = 3. Abbreviations: DMSO, dimethyl sulfoxide; HSC, hematopoietic stem cells.

Article Snippet: UCB–MNC were cultured at an empirically determined optimal density of 4.0 × 10 5 cells/ml without any cell surface marker dependent stem cell enrichment in StemSpan‐Serum Free Expansion Media (SFEM) or StemSpan‐Animal Component Free Media (ACF) (STEMCELL Technologies, Canada) supplemented with human cytokine cocktail of 100 ng/ml stem cell factor (SCF) (PeproTech, USA) and thrombopoietin (TPO) (PeproTech); 50 ng/ml FLT‐3 Ligand (FLT‐3L) (PeproTech); and 20 ng/ml insulin‐like growth factor binding protein‐2 (IGFBP‐2) (R&D Systems, USA).

Techniques: Flow Cytometry, Control, Expressing

Journal: Stem Cells Translational Medicine

Article Title: Ex Vivo Expansion of CD34 + CD90 + CD49f + Hematopoietic Stem and Progenitor Cells from Non‐Enriched Umbilical Cord Blood with Azole Compounds

doi: 10.1002/sctm.17-0251

Figure Lengend Snippet: Grafts generated by expanding UCB–MNC in presence of C7 resulted in enhanced engraftment of human cells in the PB and bone marrow (BM) of NSG mice week 2–3 of post‐transplantation. (A): Human CD45 chimerism in PB of NSG mice at week 3 post‐transplantation with non‐expanded or expanded UCB. Expansion of the UCB grafts was carried out using the mononuclear fraction (i.e., without CD34 selection) in either serum free expansion media or animal component free media that were supplemented with cytokines. The absolute cell dose of non‐expanded graft was 2.5 × 10 7 cells/kg while the expanded grafts (either fresh or frozen‐thawed) were transplanted at equivalent cell dosage of 2.5 × 10 7 cells/kg. The scatter plot represents the human CD45 chimerism of individual animals and depicts the geometric mean with 95% confidence interval (CI) of respective treatments. p values generated from Mann‐Whitney U test among indicated experimental groups are shown in the graph for the stated n values. (B): Human CD45 chimerism in PB of male NSG mice transplanted with cytokine control or C7 and cytokine expanded UCB at equivalent cell dosage of 2.5 × 10 7 cells/kg at week 6, 10, and 16 post‐transplantation. Each data point represents the geometric mean with 95% CI of respective treatments. p values generated from Mann‐Whitney U test among indicated experimental groups are shown in the graph for the stated n values. (C): Lineage commitment of the human CD45 cells those are present in the PB of NSG mice at week 3 post‐transplantation. The absolute cell dose of non‐expanded graft was 5.0 × 10 7 cells/kg while the expanded grafts were transplanted at equivalent cell dosage of 5.0 × 10 7 cells/kg. The scatter plot represents the proportion of monocytes (CD45 + CD33 + ), granulocytes (CD45 + CD15 + ), T cells (CD45 + CD3 + ), and B cells (CD45 + CD19 + ) present among the total human cells in each individual animals and depicts the geometric mean with 95% CI of respective treatments. p values generated from Mann‐Whitney U test among indicated experimental groups are shown in the graph for the stated n values. (D): Determination of severe combined immunodeficiency repopulating capacity (SRC) frequency by limiting dilution assay using L‐Calc software and Extreme Limiting Dilution Assay. A NSG mouse is considered to be positive if human CD45 chimerism is >0.40% in PB at week 3. The data is calculated at two transplantation dosage of 2.5 × 10 7 and 5.0 × 10 7 cells/kg for both male and female recipients. (E): A scatter plot of human CD45 chimerism in PB of NSG mice at week 2 post‐transplantation. The absolute cell dose of non‐expanded graft was 10.0 × 10 7 cells/kg while the expanded grafts were transplanted at equivalent cell dosage of 10.0 × 10 7 cells/kg. The scatter plot represents the human CD45 chimerism of individual animals and depicts the geometric mean with 95% CI of respective treatments. p values generated from Mann‐Whitney U test among indicated experimental groups are shown in the graph for the stated n values. (F): A scatter plot of human CD45 + CD3 + T cell chimerism in PB of NSG mice at week 2 post‐transplantation. The absolute cell dose of non‐expanded graft was 10.0 × 10 7 or 5.0 × 10 7 cells/kg while the C7 expanded grafts were transplanted at equivalent cell dosage of 10.0 × 10 7 or 5.0 × 10 7 cells/kg. The scatter plot represents the human T cell chimerism of individual animals and depicts the geometric mean with 95% CI of respective treatments. p values generated from Mann‐Whitney U test among indicated experimental groups are shown in the graph for the stated n values. (G): Kaplan‐Meier survival curve of the NSG mice transplanted with C7 or cytokine expanded UCB–MNC and non‐expanded graft over 60‐days observation period. The absolute cell dose of non‐expanded graft was 10.0 × 10 7 cells/kg while the expanded grafts were transplanted at equivalent cell dosage of 10.0 × 10 7 cells/kg. The overall statistical comparisons for the experimental groups are also shown. (H): A scatter plot of human CD45 + cells, CD45 + CD34 + progenitors, CD45 + CD3 + T cells, CD45 + CD19 + B cells and CD45 + CD34 + CD33 + myeloid progenitor cells chimerism in BM of female NSG mice at week 2 post‐transplantation. The absolute cell dose of non‐expanded graft was 10.0 × 10 7 cells/kg while the expanded grafts were transplanted at equivalent cell dosage of 10.0 × 10 7 cells/kg. The scatter plot represents the human CD45 + cells, CD45 + CD34 + progenitors, CD45 + CD3 + T cells, CD45 + CD19 + B cells, and CD45 + CD34 + CD33 + myeloid progenitor cells chimerism of individual animals and depicts the geometric mean with 95% CI of respective treatments. p values generated from Mann‐Whitney U test among indicated experimental groups are shown in the graph for the stated n values. Abbreviations: MNC, mononucleated cells; PB, peripheral blood; UCB, umbilical cord blood.

Article Snippet: UCB–MNC were cultured at an empirically determined optimal density of 4.0 × 10 5 cells/ml without any cell surface marker dependent stem cell enrichment in StemSpan‐Serum Free Expansion Media (SFEM) or StemSpan‐Animal Component Free Media (ACF) (STEMCELL Technologies, Canada) supplemented with human cytokine cocktail of 100 ng/ml stem cell factor (SCF) (PeproTech, USA) and thrombopoietin (TPO) (PeproTech); 50 ng/ml FLT‐3 Ligand (FLT‐3L) (PeproTech); and 20 ng/ml insulin‐like growth factor binding protein‐2 (IGFBP‐2) (R&D Systems, USA).

Techniques: Generated, Transplantation Assay, Selection, MANN-WHITNEY, Control, Limiting Dilution Assay, Software