scholarly journals In Vivo Analysis of PML-RARA in a Humanized Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1020-1020
Author(s):  
Hiromichi Matsushita ◽  
Takashi Yahata ◽  
Yin Sheng ◽  
Yoshihiko Nakamura ◽  
Yukari Muguruma ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Nuclear Bodies ◽  
Leukemic Cells ◽  
Human Cord Blood ◽  
Immunodeficient Mice ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. Disclosures No relevant conflicts of interest to declare.

Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 1097-1105 ◽  
Author(s):  
G. Güenechea ◽  
J.C. Segovia ◽  
B. Albella ◽  
M. Lamana ◽  
M. Ramı́rez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Immunodeficient Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P < .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

scholarly journals Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2489-2497 ◽  
Author(s):  
J Vormoor ◽  
T Lapidot ◽  
F Pflumio ◽  
G Risdon ◽  
B Patterson ◽  
...  
Keyword(s):  
Cord Blood ◽  
Dna Analysis ◽  
Hematopoietic Cells ◽  
In Vivo Model ◽  
Erythroid Progenitors ◽  
Human Cord Blood ◽  
Immunodeficient Mice ◽  
High Level

Abstract Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo- erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood- derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

scholarly journals Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2489-2497 ◽  
Author(s):  
J Vormoor ◽  
T Lapidot ◽  
F Pflumio ◽  
G Risdon ◽  
B Patterson ◽  
...  
Keyword(s):  
Cord Blood ◽  
Dna Analysis ◽  
Hematopoietic Cells ◽  
In Vivo Model ◽  
Erythroid Progenitors ◽  
Human Cord Blood ◽  
Immunodeficient Mice ◽  
High Level

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo- erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood- derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

Identification of Small Molecules Selectively Targeting Leukemic Stem Cells within Their Stromal Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 413-413
Author(s):  
Alissa R. Kahn ◽  
Kimberly A. Hartwell ◽  
Peter G. Miller ◽  
Benjamin L. Ebert ◽  
Todd R. Golub ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cord Blood ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Minimal Toxicity ◽  
Nsg Mice ◽  
Cd34 Cells

Abstract Abstract 413 Current therapies for acute myeloid leukemia (AML) are highly toxic, yet the relapse rate remains high. New therapies are needed to improve cure rates while decreasing toxicity. Because therapies may be affected by the tumor niche, we aimed to test new compounds on leukemic stem cells (LSCs) within their stromal microenvironment. A niche-based high throughput screen identified candidate small molecules potentially toxic to MLL-AF9 murine leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell et al, Blood 118, Abs 760, 2011.) Three such compounds, including a selective serotonin receptor antagonist highly specific for the 5-HT1B receptor, SB-216641, and two antihelminthics, parbendazole and methiazole, were found to be effective and selected for studies on human leukemias. We first examined SB-216641, studying the effects of this compound on 7 human primary AML samples. We began by assessing the compound's effect on LSCs using the week 5 cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. CD34+ cells were isolated with immunomagnetic beads. The leukemic cells were pulse treated for 18 hours and washed prior to placement on MS-5 murine stroma. We performed serial drug dilutions using the CAFC assay with the human primary samples as well as with HSCs derived from cord blood. All human leukemic samples formed cobblestone areas in the control setting (46-200 CAFCs/106 cells plated). IC50 for the human primary leukemia CAFCs was 630 nm, and at 10 μM all LSCs were killed while normal human HSCs had 100% survival. A combination of the AML cell line HL60 transduced with GFP-luciferase and normal cord blood CD34+ cells (1:200) were then pre-incubated overnight with SB-216641 at 5 and 10 μM and injected into Nod Scid IL2R-gamma null (NSG) mice. The control mice had leukemic engraftment by luciferase imaging and flow cytometry and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. Primary patient AML samples were also pre-incubated overnight with SB-216641 at 10 μM and injected into NSG mice. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had no engraftment following exposure to SB-216641. Finally, an in vivo study was completed on NSG mice injected intraperitoneally with 20 mg/kg/day beginning on day 1 or day 8 after inoculation with HL60 (500 cells). The mice were imaged at 2 and 3 week time points and both treatment groups had significantly less leukemia on imaging than the control group with minimal toxicity noted. Another specific 5-HT1B receptor antagonist, SB-224289, was found to have similar activity to SB-216641 against leukemic cells and to spare HSCs in preliminary studies. Similar CAFC studies with serial dilutions on primary AML samples were performed on the two anti-helminthic agents. IC50 for parbendazole was 1.25 μM and for methiazole 5 μM. As shown by luciferase imaging and flow cytometry, when injected with combined HL60 and cord blood pre-incubated overnight at 5 and 10 μM with each compound as described above, the control mice engrafted with leukemia and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. NSG mice were then injected with primary AML pretreated overnight with parbendazole at 10 μM. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had significantly lower engraftment following exposure to parbendazole (p = 0.01). Two new avenues of leukemia therapy were discovered warranting further investigation. SB-216641, an agent with a completely novel receptor target in leukemia therapy, has shown both in vitro success in human leukemia as well as preliminary success in vivo with minimal toxicity. We aim to move forward with this agent while also testing parbendazole in vivo, as this compound is already known to have good pharmacokinetics and minimal toxicity in animals. The high toxicity to LSCs and sparing of normal HSCs give both these agents an attractive profile for future clinical trials. Disclosures: Ebert: Genoptix: Consultancy; Celgene: Consultancy.

CD133 Is a Positive Marker Of Human Cord Blood-Derived CD34-Negative Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1177-1177
Author(s):  
Masaya Takahashi ◽  
Yoshikazu Matsuoka ◽  
Keisuke Sumide ◽  
Ryusuke Nakatsuka ◽  
Tatsuya Fujioka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Severe Combined Immunodeficiency ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Background We have previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). A series of our studies suggests that the identified CD34- SRCs are a distinct class of primitive hematopoietic stem cell (HSC) and that they are at the apex of human HSC hierarchy. Recently, we developed a high-resolution purification method for primitive CD34- SRCs using 18 lineage (Lin)-specific antibodies, which can enrich CD34- SRC at 1/1,000 level (Exp Hematol 2011: 39:203). In the present study, we tried to identify the positive marker of CD34- SRCs in order to further purify and characterize the CD34- SRCs (HSCs). Materials and Methods First, we extensively analyzed candidate positive markers, including known HSC markers and various adhesion molecules by FACS using highly purified CB-derived 18Lin-CD34+/- cells. Finally, we identified CD133 as a positive marker of human CB-derived CD34- SRCs. Then, CB-derived 18Lin- CD34+/-CD133+/- cells were sorted by FACS, and hematopoietic stem/progenitor cell (HSPC) capacities of these four fractions of cells were extensively investigated. HSPC capacities were evaluated using (1) colony-forming cell (CFC) assays, (2) measurement of maintenance/production of CD34+ cell capacities in co-cultures with human bone marrow-derived mesenchymal stromal cells (BM-MSCs) (Blood 2010:24:162), (3) SRC activities using NOG mice, (4) limiting dilution analyses (LDA) to determine the SRC frequency in the 18Lin-CD34-CD133+ fractions, and (5) comparison of gene expression profiles between 18Lin-CD34+/-CD133+/- cells by real-time RT-PCR. Results Seventy-five percent of 18Lin-CD34+ and 13.5% of 18Lin-CD34- cells highly expressed CD133. In the CFC assays, the plating efficiencies of 18Lin-CD34+CD133+, CD34+CD133-, CD34-CD133+ and CD34-CD133- cells were 57%, 65%, 39% and 19%, respectively. Interestingly, most of 18Lin-CD34-CD133+/- cells formed erythroid-bursts (71% and 73%) and erythro/megakaryocytes-containing mixed colonies (25% and 27%). On the contrary, they formed few granulocyte/macrophage colonies (4.2% and 0%). Then, we co-cultured these four fractions of cells with human BM-MSCs. One thousand of 18Lin-CD34+/-CD133+/- cells were seeded into each well and cells were co-cultured for 7 days in the presence of SCF+TPO+FL+IL-3+IL-6 +G-CSF. Both the 18Lin-CD34-CD133+/- cells produced CD34+ cells. However, the percentage and absolute number of CD34+ cells produced from 18Lin-CD34-CD133+ cells (31.7 % and 3.2 x 104 cells) were greater than those of 18Lin-CD34-CD133- cells (13.2 % and 0.4 x 104 cells). In addition, both the 18Lin-CD34- CD133+/- cells generated higher percentages (13.5 % and 11.5%) of CD41+ cells compared to those of the 18Lin- CD34+CD133+/- (1.8% and 4.2%) cells. Collectively, 18Lin-CD34+/-CD133+/- cells showed different in vitro lineage differentiation potentials. Then, these four fractions of cells were transplanted into NOG mice by IBMI. We performed primary and secondary transplantations for up to 36 weeks. In the results, all of the mice received 18Lin-CD34+CD133+ cells (n = 5) or 18Lin-CD34-CD133+ cells (n = 9) showed primary and secondary human CD45+ cell repopulations. However, neither 18Lin-CD34+CD133- cells nor 18Lin-CD34-CD133- cells showed human cell repopulations (n = 6 in each group). These results clearly demonstrated that the CD133 expression clearly segregated SRC activities in the 18Lin-CD34+/- cells. Moreover, LDA demonstrated that the frequency of SRCs in the 18Lin-CD34-CD133+ fraction was 1/142. Interestingly, HSC self-renewal maintenance genes, such as Notch1, HoxB4, HoxA9, and Bmi-1, were highly expressed in both 18Lin-CD34+/-CD133+ cells. Conclusion These results clearly demonstrated that CD133 is a positive marker of human CB-derived CD34- SRCs (HSCs). Furthermore, CD133 segregated SRC activities of 18Lin-CD34- as well as 18Lin-CD34+ cells in its positive fractions. More importantly, these findings suggest that number of CD133+ cells in cord blood units is a more appropriate marker to detect/predict HSC potentials in cord blood stem cell transplantation in comparison to currently used CD34+ cell numbers. Disclosures: No relevant conflicts of interest to declare.

Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 1097-1105 ◽  
Author(s):  
G. Güenechea ◽  
J.C. Segovia ◽  
B. Albella ◽  
M. Lamana ◽  
M. Ramı́rez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Immunodeficient Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P < .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

The αIIbβ3 integrin and GPIb-V-IX complex identify distinct stages in the maturation of CD34+cord blood cells to megakaryocytes

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4169-4177 ◽  
Author(s):  
Adeline Lepage ◽  
Marylène Leboeuf ◽  
Jean-Pierre Cazenave ◽  
Corinne de la Salle ◽  
François Lanza ◽  
...  
Keyword(s):  
Cord Blood ◽  
Messenger Rna ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Conditional Expression ◽  
Multistep Process ◽  
Cord Blood Cd34 ◽  
Specific Proteins ◽  
Cord Blood Cells

Abstract Megakaryocytopoiesis is a complex multistep process involving cell division, endoreplication, and maturation and resulting in the release of platelets into the blood circulation. Megakaryocytes (MK) progressively express lineage-restricted proteins, some of which play essential roles in platelet physiology. Glycoprotein (GP)Ib-V-IX (CD42) and GPIIb (CD41) are examples of MK-specific proteins having receptor properties essential for platelet adhesion and aggregation. This study defined the progressive expression of the GPIb-V-IX complex during in vitro MK maturation and compared it to that of GPIIb, an early MK marker. Human cord blood CD34+ progenitor cells were cultured in the presence of cytokines inducing megakaryocytic differentiation. GPIb-V-IX expression appeared at day 3 of culture and was strictly dependent on MK cytokine induction, whereas GPIIb was already present in immature CD34+ cells. Analysis by flow cytometry and of the messenger RNA level both showed that GPV appeared 1 day later than GPIb-IX. Microscopy studies confirmed the late appearance of GPV, which was principally localized in the cytoplasm when GPIb-IX was found on the cell surface, suggesting a delayed program of GPV synthesis and trafficking. Cell sorting studies revealed that the CD41+GPV+ population contained 4N and 8N cells at day 7, and was less effective than CD41+GPV− cells in generating burst-forming units of erythrocytes or MK colonies. This study shows that the subunits of the GPIb-V-IX complex represent unique surface markers of MK maturation. The genes coding for GPIb-IX and GPV are useful tools to study megakaryocytopoiesis and for tissue-specific or conditional expression in mature MK and platelets.

Unique Differentiation Programs of Human Fetal Liver Stem Cells Shown Both In Vitro and In Vivo in NOD/SCID Mice

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2686-2695 ◽  
Author(s):  
Franck E. Nicolini ◽  
Tessa L. Holyoake ◽  
Johanne D. Cashman ◽  
Pat P.Y. Chu ◽  
Karen Lambie ◽  
...  
Keyword(s):  
Cord Blood ◽  
Fetal Liver ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Human Fetal Liver ◽  
Human Erythropoietin ◽  
Erythroid Precursors ◽  
Lower Ratio

Comparative measurements of different types of hematopoietic progenitors present in human fetal liver, cord blood, and adult marrow showed a large (up to 250-fold), stage-specific, but lineage-unrestricted, amplification of the colony-forming cell (CFC) compartment in the fetal liver, with a higher ratio of all types of CFC to long-term culture-initiating cells (LTC-IC) and a lower ratio of total (mature) cells to CFC. Human fetal liver LTC-IC were also found to produce more CFC in LTC than cord blood or adult marrow LTC-IC, and more of the fetal liver LTC-IC–derived CFC were erythroid. Human fetal liver cells regenerated human multilineage hematopoiesis in NOD/SCID mice with the same kinetics as human cord blood and adult marrow cells, but sustained a high level of terminal erythropoiesis not seen in adult marrow-engrafted mice unless exogenous human erythropoietin (Epo) was injected. This may be due to a demonstrated 10-fold lower activity of murine versus human Epo on human cells, sufficient to distinguish between a differential Epo sensitivity of fetal and adult erythroid precursors. Examination of human LTC-IC, CFC, and erythroblasts generated either in NOD/SCID mice and/or in LTC showed the types of cells and hemoglobins produced also to reflect their ontological origin, regardless of the environment in which the erythroid precursors were generated. We suggest that ontogeny may affect the behavior of cells at many stages of hematopoietic cell differentiation through key changes in shared signaling pathways.

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