Figure 1.
Bone marrow extracts enhance hematopoietic colony formation.
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).
Figure 2.
Multilineage engraftment of human CB MNC in NSG mice.
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 3×106 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).
Figure 3.
Bone marrow extracts enhance human cord blood derived mononuclear cell engraftment in NOD/SCID mice.
Two groups of NOD/SCID mice (n = 5 each) received 3×106 CB MNCs previously cultured for 7 days, in the presence or absence of BME. A third group (n = 5) served as a negative control and were 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 and lymphoid populations to total human leukocyte engraftment was determined using CD33, CD19 and CD45 markers, respectively. *: p<0.05.
Figure 4.
Bone marrow extracts enhance human cord blood derived mononuclear cell engraftment in NSG mice.
Two groups of NSG mice (n = 4 each) received 1.5×106 CB MNCs previously cultured for 7 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 and lymphoid populations to total human leukocyte engraftment was determined using CD33, CD19 and CD45 markers, respectively. *: p<0.05.
Figure 5.
Bone marrow extracts enhance human erythroid engraftment in NSG mice.
Two groups of NSG mice (n = 3 each) received 4×106 CB MNCs previously cultured for 7 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 three 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.
Figure 6.
Bone marrow extracts enhance human bone marrow derived CD34 positive cells engraftment in immunodeficient mice.
Two groups of NSG mice (n = 5 each) received 50×103 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.