Journal: Stem Cell Investigation

Article Title: Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation

doi: 10.21037/sci-2022-026

Figure Lengend Snippet: Confirmation of creation of bone marrow derived CD34 + HHCC via PEG-mediated ex vivo cell fusion procedure. (A) Study design of the creation process of a new HHCC cell line from bone marrow aspirates of the two unrelated human donors, the male and the female donor. (B) Confirmation of HHCC ex vivo fusion assessed by FACS. Representative dot-plots of the fusion procedure: first plot on the left shows cell population before sorting (gate R1), second plot from the left side presents PKH67 staining (gate R2), next panel indicates PKH26 staining (gate R4) and the last plot on the right shows double stained population of the created HHCC after FACS sorting in gate R3 (fusion efficacy 48.29%). (C) Representative immunofluorescence images of CD34 + parent cells from two unrelated human bone marrow donors stained with PKH67 (green) and PKH26 (red) membrane dyes: left: CD34 + from the male donor (green), middle: CD34 + from the female donor (red), the right image: represents the created HHCC (yellow) by overlapping fluorescence images of PKH26/PKH67 dyes confirming the chimeric state of HHCC. Magnification: ×65. (D) Representative images of HHCC from electron microscopy (JEOL, JEM 1220, Tokyo, Japan). On the left image CD34 + parent cell prior to cell fusion, on the right image of HHCC at 24 hours after parent cell fusion where two nuclei are visible. Scale bar: 5.0 µm. HHCC, human hematopoietic chimeric cells; PEG, polyethylene glycol; FACS, fluorescence activated cytometry sorting; PKH, Paul Karl Horan dyes; SSC, side scatter; FSC, forward scatter.

Article Snippet: The best efficiency of cell propagation was achieved using the StemSpan TM H3000 (StemCell Technologies) medium supplemented with the StemSpan TM CD34 + expansion supplement.

Techniques: Derivative Assay, Ex Vivo, Staining, Immunofluorescence, Membrane, Fluorescence, Electron Microscopy, Cytometry

Journal: Stem Cell Investigation

Article Title: Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation

doi: 10.21037/sci-2022-026

Figure Lengend Snippet: Confirmation of propagation and proliferation of HHCC in vitro over 28 days period after cell fusion. (A) Representative images of HHCC taken during the cell propagation process at 0, 7, 14, 21 and 28 days after the fusion. Images taken from cell culture under an inverted optical microscope (Leica, magnification ×40). (B) Assessment of HHCC proliferative properties determined by comparison between the numbers of proliferating unfused CD34 + cells and fused CD34 + cells (HHCC) at 0, 7, 14, 21 and 28 days after cell fusion, assessed by Absolute Counting Beads. The experiment was repeated three times (n=3). Data are shown as mean ± SD. HHCC, human hematopoietic chimeric cells; SD, standard deviation.

Article Snippet: The best efficiency of cell propagation was achieved using the StemSpan TM H3000 (StemCell Technologies) medium supplemented with the StemSpan TM CD34 + expansion supplement.

Techniques: In Vitro, Cell Culture, Microscopy, Comparison, Standard Deviation

Journal: Stem Cell Investigation

Article Title: Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation

doi: 10.21037/sci-2022-026

Figure Lengend Snippet: Confirmation of high viability and low apoptosis level in the created HHCC up to 21 days after cell fusion. (A) Viability assessment of created HHCC: upper panel, the positive control—CD34 + cells treated with 500 µM of H 2 O 2 , middle panel, the parent viable CD34 + cells—the viable control and lower panel, the fused CD34 + (HHCC) cells, assessed by Trypan Blue at 3 hours and 7 days after fusion, visualized by light microscopy, Magnification ×40. (B) Comparative analysis of viability of the fused CD34 + (HHCC) and unfused viable parent CD34 + cells measured at 0, 7, 14 and 21 days after cell fusion and assessed by Trypan Blue staining confirming high, over 98% viability for both, the fused CD34 + (HHCC) cells and the unfused viable CD34 + controls. (C) Assessment of HHCC viability by LIVE/DEAD ® assay by flow cytometry revealed 98.66% viability at 3 hours, 98.37% at 4 days and 98.24% viability at 7 days after fusion confirming safety of fusion procedure. (D) Evaluation of fused CD34 + cells (HHCC) for early signs of apoptosis by Annexin V-APC antibody and for the presence of dead cells by Sytox Blue staining. Analysis by flow cytometry after cell fusion revealed: at 3 hours after fusion—2.72% of HHCC in the early stage of apoptosis and 1.31% of dead cells, and at 4 days after fusion—3.2% of HHCC in the early stage of apoptosis and 0.57% of dead cells, and at 7 days after fusion, 2.52% of HHCC in the early stage of apoptosis and 0.81% of dead cells. (E) Assessment of HHCC apoptosis (fused CD34 + cells) in cell culture by TUNEL assay for detection of the late stage of apoptosis (nuclear degradation) by flow cytometry, revealed 1.23% of apoptotic cells at 3 hours after fusion and 2.19% apoptotic cells at 7 days after fusion. (F) Comparative analysis of cellular apoptosis by TUNEL assay in the cell culture of fused CD34 + (HHCC) and unfused CD34 + donor cells revealed low percentage (3.2–3.9%) of apoptotic cells in both groups up to 21 days after fusion procedure. HHCC, human hematopoietic chimeric cells.

Article Snippet: The best efficiency of cell propagation was achieved using the StemSpan TM H3000 (StemCell Technologies) medium supplemented with the StemSpan TM CD34 + expansion supplement.

Techniques: Positive Control, Control, Light Microscopy, Staining, Live Dead Assay, Flow Cytometry, Cell Culture, TUNEL Assay

Journal: Stem Cell Investigation

Article Title: Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation

doi: 10.21037/sci-2022-026

Figure Lengend Snippet: Confirmation of the hematopoietic phenotype stability of the created HHCC cell line after cell fusion by expression of the hematopoietic cell surface markers. (A) Representative flow cytometry histograms of the hematopoietic and stem cell surface markers (CD34 + , CD133 + , CD117 + , CD90 + , CD4 + , CD19 + , CD14 + ) in the created HHCC at 3 and 24 hours after fusion procedure. (B) Expression of the hematopoietic cell surface markers (CD34 + , CD133 + , CD117 + , CD90 + , CD4 + , CD19 + , CD14 + ) assessed at 3 and 24 hours after cell fusion. Increased expression of CD133 + , CD4 + and CD14 + , observed at 24 hours after cell fusion. (C) Expression of the stem and progenitors’ cells surface markers including: precursor stem cells (CD133 + /CD90 + ), T-cells (CD4 + /CD8 + ), T-regulatory cells (CD4 + /CD25 + ), B-cells (CD5 + /CD19+), and dendritic cells (CD123 + /CD11c + ) evaluated at 3 hours and 7 days of cell culture after fusion. Increased expression of CD4 + /CD8 + , CD4 + /CD25 + , CD5 + /CD19 + and CD123 + /CD11c + cells was observed at 7 days after cell fusion. (D) Representative flow cytometry dotplots of T-regulatory cells surface markers (CD4 + /CD25 + ) assessed in the CD34 + naive donor cells (upper row) and in the created HHCC (lower row) at 7 days and 21 days of cell culturing after fusion procedure. HHCC, human hematopoietic chimeric cells.

Article Snippet: The best efficiency of cell propagation was achieved using the StemSpan TM H3000 (StemCell Technologies) medium supplemented with the StemSpan TM CD34 + expansion supplement.

Techniques: Expressing, Flow Cytometry, Cell Culture

Journal: Stem Cell Investigation

Article Title: Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation

doi: 10.21037/sci-2022-026

Figure Lengend Snippet: Confirmation of the genotype of the HHCC created from the two unrelated human CD34 + donors. (A) Representative immunofluorescence images of the CD34 + cells from two unrelated donors (male, the left image and female, the right image) and (B) assessment of HHCC at 3 hours after fusion by FISH, revealed a low level of 0.48% of the polyploid cells in the HHCC population. Probes were stained with specific X (red) and Y-chromosome specific (green) dyes, counterstained with DAPI and analyzed by confocal microscopy. The presence of at least three X and one Y chromosomes was considered a polyploidy (original magnification, ×60). (C) PCR-rSSOP analysis of HHCC confirming presence of the parent cells HLA class I and class II antigens specific for both CD34 + donors—the male (blue) and the female (red) donor at 3 hours and 7 days after cell fusion. (D) STR-PCR analysis validating genetic stability of HHCC cells determined by the presence of all STR loci derived from both CD34 + donors—the male (blue) and the female (red) donor at 3 hours and at 7 days after cell fusion. HHCC, human hematopoietic chimeric cells; HLA, human leukocyte antigen; FISH, fluorescence in situ hybridization; STR, short tandem repeats; PCR, polymerase chain reaction; rSSOP, reverse sequence-specific oligonucleotide probe.

Article Snippet: The best efficiency of cell propagation was achieved using the StemSpan TM H3000 (StemCell Technologies) medium supplemented with the StemSpan TM CD34 + expansion supplement.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Derivative Assay, Fluorescence, In Situ Hybridization, Polymerase Chain Reaction, Sequencing

Journal: Stem Cell Investigation

Article Title: Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation

doi: 10.21037/sci-2022-026

Figure Lengend Snippet: Confirmation of clonogenic (A,B) and immunomodulatory properties (C,D) of the created HHCC. (A,B) Clonogenic properties of HHCC assessed by CFU assay. (A) Representative images of CFU-GM, BFU-E and CFU-GEMM colonies taken under an inverted optical microscope (magnification ×40) confirmed clonogenic properties of the created HHCC cell line at 3 hours after fusion. (B) Assessment of the number of the CFU-GM, BFU-E and CFU-GEMM colonies in the unstained CD34 + cells before fusion, in the fused CD34 + (HHCC) cells and in the PKH fluorescently labeled CD34 + cells, at 3 hours (left graph) and 7 days (right graph) after fusion. Over the time of cell’s propagation, the number of CFU-GM colonies increased at the expense of BFU-E and CFU-GEMM colonies. (C,D) Tolerogenic potential of HHCC assessed by MLR. (C) Representative histograms of flow cytometry analysis by MLR assay. On the left, the histogram of positive control presenting the percentage of proliferative lymphocytes (responder T-cells—25.3%) after 4 days of cell culture with 3rd party irradiated allogeneic T-cells. On the right, histogram presents the percentage of proliferating responders T-cells—14.1% after 4 days of cell culture with fused CD34 + cells (HHCC). The number of proliferative lymphocytes (responder CD8 + T-cells) in the positive control was higher than in the sample containing fused cells (HHCC). (D) Real-time PCR analysis of the expression of mRNA for pro-tolerogenic Th2 (IL-10, TGF-β) and pro-inflammatory Th1 (TNF-α) cytokines of CD34 + cells before fusion, and fused HHCC at 3 hours, 7 days, and 14 days after fusion procedure. Assessment of cell’s propagation over time revealed significantly increased expression of the pro-tolerogenic cytokines (IL-10). Data are presented as mean ± SD, ****, P<0.0001. HHCC, human hematopoietic chimeric cells; CFU, colony forming units; GM, granulocyte macrophage; BFU, burst forming unit; E, erythroid; GEMM, granulocyte, erythroid, macrophage, megakaryocyte; MLR, mixed lymphocyte reaction; PKH, Paul Karl Horan dyes; PCR, polymerase chain reaction; IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor; SD, standard deviation.

Article Snippet: The best efficiency of cell propagation was achieved using the StemSpan TM H3000 (StemCell Technologies) medium supplemented with the StemSpan TM CD34 + expansion supplement.

Techniques: Colony-forming Unit Assay, Microscopy, Labeling, Flow Cytometry, Mlr Assay, Positive Control, Cell Culture, Irradiation, Real-time Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Standard Deviation