Journal: PLoS ONE

Article Title: Acellular Bone Marrow Extracts Significantly Enhance Engraftment Levels of Human Hematopoietic Stem Cells in Mouse Xeno-Transplantation Models

doi: 10.1371/journal.pone.0040140

Figure Lengend Snippet: A ) About 20,000 CB MNCs from each patient were plated on methylcellulose-based media (HSC002, HSC003, and HSC004), in the presence or absence of BME (CB+BME or CB, respectively). Methylcellulose media contained no growth factors (HSC002), SCF+GM-CSF+IL3+Epo (HSC003), or SCF+GM-CSF+IL3 without Epo (HSC004). Colony forming units (CFU) consisting of CFU-G/M (Granulocyte or Macrophage or both), CFU-E/BFU-E (Erythroid), and CFU-GEMM (Granulocyte Erythroid Macrophage Megakaryocyte) were counted at day 16 using an inverted microscope. Data represents an average of 6 different samples. B ) A total of 100 CB CD34+ cells from each patient were plated on methylcellulose media (HSC002, HSC003, and HSC004), in the presence or absence of BME (CB+BME or CB, respectively) as described above. Data represents an average of 4 samples used in the study. Paired TTest was used for statistical significance (*: p<0.05, **: p<0.001).

Article Snippet: On the other hand, about 20,000 CD34 + cells were seeded in 6-well plates and suspended at 10 5 cells/mL in serum-free StemSpan H3000 culture medium (Stem Cell Technologies).

Techniques: Inverted Microscopy

Journal: PLoS ONE

Article Title: Acellular Bone Marrow Extracts Significantly Enhance Engraftment Levels of Human Hematopoietic Stem Cells in Mouse Xeno-Transplantation Models

doi: 10.1371/journal.pone.0040140

Figure Lengend Snippet: In this study, NOD/SCID or NSG mice received iv busulfan conditioning followed by iv injection of CB MNC or CD34 + cells that were previously cultured in vitro for 7 days, in the presence or absence of BME. This figure represents NSG mice which received iv injection of 3×10 6 CB MNC according to the same protocol. Mice were sacrificed and bone marrow cells were harvested from femurs, tibia and pelvis and examined for multilineage engraftment by flow cytometry according to the following gating strategy: A ) Live cells were first gated using forward scatter versus side scatter plots (R1 region). The three plots represent mice injected with saline (negative control), CB, or CB+BME; respectively. B ) Human leucocytes were then gated using human CD45 staining (pan-leukocyte marker, R2 region). C ) From CD45 + gate (R2 region), cells were then examined for multi-lineage engraftment defined by the presence of separate lymphoid (CD45 + CD19 + , R3 region) and myeloid (CD45 + CD33 + , R4 region) populations. D ) The population of CD45 − cells were gated in order to determine the erythroid populations. Indeed, erythroid populations made up of mature RBCs (CD45 − CD36 − CD235a + , R5 region) or immature erythroblasts (CD45 − CD36 + CD235a + , R6 region) were also determined. It’s important to note that the CD36 + CD235a − population, which is not present in the controls, does not represent immature erythroblasts but are CD45+ mature cells. The percent engraftment was defined as the total number of leucocytes and immature erythroblasts (CD45 + and CD45 − CD36 + CD235a + cells).

Article Snippet: On the other hand, about 20,000 CD34 + cells were seeded in 6-well plates and suspended at 10 5 cells/mL in serum-free StemSpan H3000 culture medium (Stem Cell Technologies).

Techniques: IV Injection, Cell Culture, In Vitro, Flow Cytometry, Injection, Saline, Negative Control, Staining, Marker

Journal: PLoS ONE

Article Title: Acellular Bone Marrow Extracts Significantly Enhance Engraftment Levels of Human Hematopoietic Stem Cells in Mouse Xeno-Transplantation Models

doi: 10.1371/journal.pone.0040140

Figure Lengend Snippet: Two groups of NSG mice (n = 5 each) received 50×10 3 bone marrow derived CD34 positive cells previously cultured for 13 days, in the presence or absence of BME. A third group (n = 5) served as a negative control and was injected with saline. Mice were sacrificed six weeks after transplantation. A ) The percentage of engraftment was determined using the human pan leukocyte CD45 marker. B ) The contribution of myeloid, lymphoid and erythroid populations to total human leukocyte engraftment was determined using CD33, CD19, CD36 and CD45 markers, respectively. *: p<0.05.

Article Snippet: On the other hand, about 20,000 CD34 + cells were seeded in 6-well plates and suspended at 10 5 cells/mL in serum-free StemSpan H3000 culture medium (Stem Cell Technologies).

Techniques: Derivative Assay, Cell Culture, Negative Control, Injection, Saline, Transplantation Assay, Marker

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In vitro and in vivo expansion of CD33/HBG promoter-edited HSPCs with Mylotarg

doi: 10.1016/j.omtm.2024.101343

Figure Lengend Snippet: Evaluation of HDAd-ABE8e-sgHBG#2-sgCD33 ΔE2 transduction in CD34 + cells (A) Experimental procedure. CD34 + cells from healthy donors ( N = 4 CD34 + cells donors, in duplicate in the transduced group) were cultured in medium supplemented with low cytokines and a combination of the small molecules, SR1 and Ly, to preserve their stemness during the culture. After recovery from cryopreservation, the cells were transduced with the HDAd-ABE8e-sgHBG#2-sgCD33 ΔE2 vector at an MOI of 4,000 vp/cell and cultured for a total of 10 days. (B) Percentage of target base conversion by HDAd-ABE8e-sgHBG#2-sgCD33 ΔE2 at both CD33 and HBG sites 72 h post transduction ( N = 4). NGS data were analyzed by CRISPResso2. (C) Expression of CD33 3 and 10 days post transduction compared with untransduced CD34 + cells. CD33 FL is the unedited CD33 version ( N = 4 CD34 + cell donors, in duplicate in the transduced group). CD33 ΔE2 is the exon 2 deleted version. (D) Representative histograms showing the percentage of CD33 levels on day 10. (Ε) Frequency of CD34 + /CD38 – cells on day 10 ( N = 4 CD34 + cells donors, in duplicate in the transduced group). (F) Frequency of CD34 + /CD38 – /CD90 + cells on day 10 ( N = 4 CD34 + cell donors, in duplicate in the transduced group). (G) Number of total erythroid (BFU-Es), and myeloid (CFU-GM) colonies per 3,000 plated CD34 + cells ( N = 4). Data are shown as means ± SEM. ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, ∗ p ≤ 0.05 (two-way ANOVA with Bonferroni correction).

Article Snippet: The cells were recovered from frozen stocks and incubated for 24 h in serum free medium (Stemspan H3000, STEMCELL Technologies) supplemented with penicillin/streptomycin (Gibco), Flt3 ligand (100 ng/mL), thrombopoietin (100 ng/mL), and stem cell factor (SCF) (100 ng/mL) and the small molecules SR1 (1 μΜ) (Cellagen Technology) and Ly2228820 (Ly, 100 nM) (Selleckchem)., , All cytokines were obtained from PeproTech.

Techniques: Transduction, Cell Culture, Plasmid Preparation, Expressing