Role of Integrin Linked Kinase in Expansion and Chemotaxis of CD34+ Cord Blood Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3616-3616
Author(s):  
Sunanda Basu ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Growth Factors ◽  
Growth Factor ◽  
Ex Vivo ◽  
Β1 Integrin ◽  
Cd34 Cells ◽  
Integrin Linked Kinase ◽  
In Cells

Abstract 3616 Poster Board III-552 Despite being an excellent alternative to bone marrow or mobilized peripheral blood as a source of hematopoietic stem progenitor cells (HSPCs), the limiting factor to wider use of cord blood (CB) in transplantation is the 10-fold lower cell dose in a typical CB unit compared to harvested bone marrow or peripheral blood stem cells. Successful ex-vivo expansion of HSPCs as well as increasing transplantation efficiency by adopting protocols that enhance homing and engraftment of transplanted HSPC provides hope of making the applicability of a single unit of CB wider in the adult population. Interaction and adhesion of HSPCs with extracellular matrix (ECM) is an important event that regulates cell differentiation, proliferation, survival/ apoptosis as well as migration of HSPCs. Based on evidence present in the literature it appears that in addition to cooperative action between adhesion and growth factors, integrin mediated cell adhesion also provides distinct growth regulatory cues. Although it is known that in hematopoietic system, β1 integrin plays an important role in the interaction of HSPCs with integrin ligands, the underlying molecular mechanisms regulating βl integrin activity in hematopoietic cells remains largely unknown. Based on studies in other systems- Integrin linked kinase (ILK) appears to be an important molecule. ILK not only modulates β1 integrin activity, but also functions as an adapter protein, physically coupling downstream signals from both integrins and growth factors. ILK is preferentially expressed in murine stem / early progenitor cells (based on I. Lemiscka's Stem cell database) and we have found that ILK is expressed in both primitive (CD38-/loCD34+) and committed progenitor (CD38+ CD34+) cells from CB by western blot. Moreover, using a co-immunoprecipitation approach, we found that in freshly isolated CD34+CB cells ILK interacts with β1 integrin. To investigate the role of ILK in adhesion-dependent and -independent growth of CB CD34+ cells both in the presence and absence of growth factors we expressed constitutive active ILK (CAILK) or dominant negative ILK (DNILK) in CD34+CB cells. In addition, we have also evaluated the effect of modulating ILK activity on chemotaxis of CD34+ CB cells towards stromal derived factor-1 (SDF-1)/CXCL12. We found that expression of CAILK enhances expansion of total CD34+ cells as well as colony forming cells stimulated ex-vivo by growth factors (stem cell factor- SCF; Flt-3 ligand and thrombopoietin-TPO), compared to cells transfected with vector alone. In contrast, expression of DNILK inhibited expansion of CD34+CB cells; this effect was more pronounced when cells were cultured in the absence of fibronectin, the ECM. Expression of CAILK increases cell-cycling since a greater proportion of cells were in ‘S’-phase compared to cells expressing DNILK or vector alone, both when the cells were expanded in the presence or absence of fibronectin for 20h. Expression of CAILK also leads to improved survival of CD34+ cells in the absence of serum and growth factors. Interestingly, β-catenin could be detected in CD34+ cells expressing CAILK, but not in cells transfected with DNILK or vector alone. Furthermore, in response to growth factor stimulation, Akt is phosphorylated in cells expressing either CAILK or DNILK; however, the amount of Akt phosphorylation was lower in cells expressing DNILK. Expression of CAILK improved modestly but not significantly the chemotaxis of CD34+ cells towards SDF-1 compared to cells expressing vector alone. However, DNILK significantly impaired chemotaxis of CD34+CB cells towards SDF-1. This impairment of chemotaxis is associated with defective actin polymerization in response to SDF-1, both at the ‘leading-edge’ and ‘tail’ of a polarized DNILK expressing CD34+ cell. Our findings implicate a role for ILK in both growth factor stimulated ex-vivo expansion of HSPCs as well as SDF-1 mediated chemotaxis. This may have potential implications in the therapeutic use of CB cells. Disclosures: No relevant conflicts of interest to declare.

The Ex-Vivo Expansion of Megakaryocytic Progenitors from Hematopoietic Stem Cells for Thrombocytopenia in NOD/SCID Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3746-3746
Author(s):  
Mo Yang ◽  
Jieyu Ye ◽  
Enyu Liang ◽  
Chunfu Li ◽  
Beng H Chong
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Growth Factor ◽  
Cord Blood ◽  
Ex Vivo ◽  
Cell Types ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Objective: Thrombocytopenia is a common clinical problem in patients with cancer or bone marrow transplantation. Currently it is mainly managed by platelet transfusion. Repeated platelet transfusions are associated with the risks of transfusion reactions/alloimmunisation and may lead to platelet refractoriness. Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is other strategy for the treatment of thrombocytopenia. This study aimed to establish efficient conditions for the expansion of the MK progenitors from enriched CD34(+) cells of umbilical cord blood. Methods: This study investigated the effect of flt-3 ligang (FL), stem cell factor (SCF) and platelet-derived growth factor (PDGF) in combination with other megakaryocyte-promoting cytokines such as thrombopoietin (TPO) on the differentiation and proliferation of megakaryocytic progenitors. As an early acting growth factor, FL may promote the ex vivo expansion of hematopoietic stem and progenitor cells. We compared the effects of FL and SCF in combination with other megakaryocyte-promoting cytokines in megakaryocytic progenitors. Results: In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus TPO, interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the effect of FL in augmenting the expansion of MK progenitors might be due to the early action of FL at the pluripotent stem cell stage. Our results also demonstrated that TPO alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of in IL-1 beta, FL and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and FL. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice. Conclusions: This study showed that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications. Disclosures Yang: National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

scholarly journals Paracrine Mechanisms of Intravenous Bone Marrow-Derived Mononuclear Stem Cells in Chronic Ischemic Stroke

2016 ◽  
Vol 6 (3) ◽  
pp. 107-119 ◽  
Author(s):  
Ashu Bhasin ◽  
M.V. Padma Srivastava ◽  
Sujata Mohanty ◽  
Sivasubramaniam Vivekanandhan ◽  
Sakshi Sharma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Ischemic Stroke ◽  
Growth Factors ◽  
Growth Factor ◽  
Stroke Patients ◽  
Bdnf Expression ◽  
Serious Adverse Events ◽  
Significant Difference

Background: The emerging role of stem cell technology and transplantation has helped scientists to study their potential role in neural repair and regeneration. The fate of stem cells is determined by their niche, consisting of surrounding cells and the secreted trophic growth factors. This interim report evaluates the safety, feasibility and efficacy (if any) of bone marrow-derived mononuclear stem cells (BM-MNC) in chronic ischemic stroke by studying the release of serum vascular endothelial growth factor (VEGF) and brain-derived neurotrophic growth factor (BDNF). Methods: Twenty stroke patients and 20 age-matched healthy controls were recruited with the following inclusion criteria: 3 months to 1.5 years from the index event, Medical Research Council (MRC) grade of hand muscles of at least 2, Brunnstrom stage 2-5, conscious, and comprehendible. They were randomized to one group receiving autologous BM-MNC (mean 60-70 million) and to another group receiving saline infusion (placebo). All patients were administered a neuromotor rehabilitation regime for 8 weeks. Clinical assessments [Fugl Meyer scale (FM), modified Barthel index (mBI), MRC grade, Ashworth tone scale] were carried out and serum VEGF and BDNF levels were assessed at baseline and at 8 weeks. Results: No serious adverse events were observed during the study. There was no statistically significant clinical improvement between the groups (FM: 95% CI 15.2-5.35, p = 0.25; mBI: 95% CI 14.3-4.5, p = 0.31). VEGF and BDNF expression was found to be greater in group 1 compared to group 2 (VEGF: 442.1 vs. 400.3 pg/ml, p = 0.67; BDNF: 21.3 vs. 19.5 ng/ml) without any statistically significant difference. Conclusion: Autologous mononuclear stem cell infusion is safe and tolerable by chronic ischemic stroke patients. The released growth factors (VEGF and BDNF) in the microenvironment could be due to the paracrine hypothesis of stem cell niche and neurorehabilitation regime.

scholarly journals Characterization of Monoclonal Antibodies That Recognize Canine CD34

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1977-1986 ◽  
Author(s):  
Peter A. McSweeney ◽  
Katherine A. Rouleau ◽  
Philip M. Wallace ◽  
Benedetto Bruno ◽  
Robert G. Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Stem Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34− cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit–granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34− cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of ≤1.1 × 107 /kg that were 29% to 70% CD34+. Engraftment kinetics were similar to those of dogs previously transplanted with approximately 10- to 100-fold more unmodified autologous marrow cells. This suggests that CD34+ is a marker not only of canine bone marrow progenitors but also for cells with radioprotective or marrow repopulating function in vivo. MoAbs to CD34 will be valuable for future studies of canine hematopoiesis and preclinical studies concerning stem cell transplantation, gene therapy, and ex vivo progenitor cell expansion.

Abstract WP146: Intravenous Bone Marrow Derived Mononuclear Stem Cells in Chronic Ischemic Stroke-Paracrine Mechanisms of Recovery

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Ashu Bhasin ◽  
Padma Srivastava ◽  
Sujata mohanty ◽  
Vivekanandhan Subramaniyam ◽  
Senthil kumaran ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Ischemic Stroke ◽  
Growth Factors ◽  
Growth Factor ◽  
Control Groups ◽  
Neural Repair ◽  
Traditional Concept ◽  
And Control

Background: The emerging role of Stem cell technology and transplantation has helped scientists to study its potential role in neural repair and regeneration. The fate of stem cells is determined by its niche, consisting of surrounding cells and the secreted trophic growth factors. This present study evaluates the safety, feasibility and efficacy of bone marrow derived mononuclear stem cells (BM-MNC) in chronic ischemic stroke by studying the release of serum vascular endothelial growth factor (VEGF) and brain derived neurotrophic growth factor (BDNF). Methods: Twenty (n=20) stroke patients recruited with the inclusion criteria as: 3 months to 1.5 years of index event, power of hand muscles atleast 2; Brunnstrom stage: 2-5; conscious and comprehendible, were randomized to study and control groups receiving autologous mean 60-70 million BM-MNC and controls receiving neuromotor rehabilitation regime only for 8 weeks. Clinical assessment (FM, mBI, MRC, Ashworth) and serum VEGF & BDNF were done at baseline and 8 weeks (2 months). Results: No patients exhibited any complication or adverse events during the whole procedure. There was no statistical significant clinical improvement between study and control groups (FM: 95%CI; 15.2 to 5.35, p=0.25; mBI : 95% CI;14.3 to 4.5, p=0.31). VEGF and BDNF were increased after stem cell infusion and exercise training in both the groups (VEGF: 442.1 pg/ml vs 400.3pg/ml; p=0.67; BDNF:95% CI∼15.09 to 4.09, p=0.57), without any statistical significant result. Conclusion: Autologous mononuclear stem cell infusion is safe and tolerable by chronic ischemic stroke. The paracrine hypothesis extends the traditional concept of stem cell niche to study the influence of stem cell released growth factors (VEGF and BDNF) on the microenvironment.

Ex-Vivo Expansion of Megakaryocyte Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2885-2885
Author(s):  
Antonio Giulivi ◽  
Mike Halpenny ◽  
Paul Birch ◽  
Lin Yang ◽  
Lisa Martin
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplant ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Marrow Transplant ◽  
Hematopoietic Progenitor Cell ◽  
Transplant Patients ◽  
Cd34 Cells ◽  
Free Media

Abstract Following Bone Marrow Transplant, thrombocytopenia and neutropenia always occur and patients require additional post transplant support in the form of platelet transfusions. Megakaryocytes (Mk), the precursors of platelets, are contained in hematopoietic progenitor cell products but their number is variable and relatively low. The infusion of ex vivo expanded Mk precursors could be beneficial by shortening the time to platelet engraftment and therefore reducing the amount of platelet transfusion support required by bone marrow transplant patients. The objective of this project was to investigate the expansion of Mk progenitors from peripheral blood stem cell (PBSC) harvests from patients with haematological malignancies. Briefly, CD34+ cells were isolated and cultured in serum free media supplemented with thrombopoietin (TPO) and Interleukin 1 (IL-1) then incubated at 37°C /5% CO2 for 8 – 12 days. Megakaryocyte progenitor analysis was accomplished using flow cytometry analysis (CD34+/41+, CD41+, CD61+) and Mk culture analysis (CFU-Mk) (Stem Cell Technologies). Mk progenitor expansion efficiency was determined as “fold expansion” of Mk progenitors produced over input levels. After 8 days of culture, a mean expansion of 46 fold (range 1.2 – 327.0, n=10) in megakaryocytic cells (CD61+) and a 15 fold expansion (1.2 – 41.7, n=10) in megakaryocyte progenitor cells (CD34+/41+) was observed. After 12 days, a 116 fold expansion (1.5 – 286, n=7) in megakaryocytic cells (CD61+) and a 19 fold expansion (2.4 – 40, n=7) in Mk progenitors (CD34+/CD41+) was observed. This study demonstrates that CD34+ cells can be used to effectively expand megakaryocytic cells using just two cytokines for an incubation period of 8 – 12 days. This data could be used to develop future protocols for use in clinical applications.

Viability in Late Stages of Ex Vivo Erythropoiesis Is Enhanced by Increased Cell Density

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4748-4748
Author(s):  
Daniela Boehm ◽  
Mohamed Al-Rubeai ◽  
William G Murphy
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Red Blood Cells ◽  
Cell Density ◽  
Late Stage ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Ex Vivo ◽  
Cd34 Cells ◽  
Late Stages

Abstract Erythropoiesis is one of the body’s most productive cell production processes yielding 2×1011 new red cells from hematopoietic stem cells (HSCs) of the bone marrow every day. Intensive research has focused on mimicking this process ex vivo through application of various growth factor combinations or co-culture with stromal cells. To develop a scalable and reproducible system for large scale production of red blood cells we have investigated in vitro erythropoiesis of peripheral blood derived CD34+ cells with primary focus on the impact of the microenvironment on the process. The influence of cultivation conditions on expansion of erythroid progenitor cells and their terminal differentiation to mature red blood cells were studied in stroma-free liquid culture supplemented with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO). Peripheral blood derived CD34+ cells were expanded by more than 105 fold over a 3 week period. This degree of expansion has only been achieved previously for CD34+ cells derived from more potent stem cell sources such as cord blood, bone marrow and G-CSF mobilized peripheral blood (Giarratana et al, Nat Biotechnol 2005). The natural environment of human erythropoiesis, the bone marrow, is a very crowded milieu where hematopoietic precursors and other cells are packed in close proximity. Cell crowdedness was found to have significant influences on ex vivo erythropoiesis. Cell density per surface area rather than cell concentration per media volume determined cell expansion during exponential growth where more crowded cells showed reduced overall expansion. In cultures inoculated at 4×105 cells/ml (2.1×105 cells/cm2) increasing cell density per area (i.e. decreasing surface area to volume ratio) 4fold (to 8.4×105 cells/cm2) resulted in 35±12% reduction of total expansion (p<0.05, unpaired Student’s t-test). While 4fold increase of cell density in cultures seeded at 1×106 cells/ml (from 5.3×105 cells/cm2 to 2.1×106 cells/cm2) reduced overall expansion by 51±9% (p<0.01). In late stage erythropoiesis, however, when cells had become arrested in G1 and no longer proliferated, cell density was seen to enhance cell viability. Dilution series of late stage erythroblasts showed that although cell viability gradually decreased over a 14 day cultivation period the decreasing rate was lower in cells cultivated at higher density as shown in the Figure. Enhanced viability in crowded culture conditions could reflect the cells’ dependency on direct cell-cell interactions as found in the marrow environment. Cultures grown to high cell densities of 2–3×106 cells/cm2 showed higher maturation efficiency than previously obtained in this cultivation set-up with more than 80% of cells being CD71-/GpA+. Enucleation yields of up to 45% were achieved indicating a significant amount of terminal maturation to red blood cells. Efficient maturation and particularly enucleation have in many cases been found to be dependent on or improved by interactions with feeder cells or macrophages (Fujimi et al, Int J Hematol 2008). Keeping erythroid cells at high densities during late stages of erythropoiesis possibly helps to mimic their in vivo environment, thus allowing for better survival and efficient terminal maturation without the need for co-culture with other cells. Figure Figure

scholarly journals Characterization of Monoclonal Antibodies That Recognize Canine CD34

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1977-1986 ◽  
Author(s):  
Peter A. McSweeney ◽  
Katherine A. Rouleau ◽  
Philip M. Wallace ◽  
Benedetto Bruno ◽  
Robert G. Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Stem Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Cd34 Cells ◽  
Marrow Cells

Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34− cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit–granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34− cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of ≤1.1 × 107 /kg that were 29% to 70% CD34+. Engraftment kinetics were similar to those of dogs previously transplanted with approximately 10- to 100-fold more unmodified autologous marrow cells. This suggests that CD34+ is a marker not only of canine bone marrow progenitors but also for cells with radioprotective or marrow repopulating function in vivo. MoAbs to CD34 will be valuable for future studies of canine hematopoiesis and preclinical studies concerning stem cell transplantation, gene therapy, and ex vivo progenitor cell expansion.

scholarly journals Hepatoma-Derived Growth Factor Is a Novel Factor to Promote the Proliferation of Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1471-1471
Author(s):  
Munetada Haruyama ◽  
Kozo Yamaichi ◽  
Akira Niwa ◽  
Megumu K Saito ◽  
Tatsutoshi Nakahata
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Absolute Number ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
The Mean

Abstract Ex vivo expansion of hematopoietic stem cells (HSCs) is an attractive therapeutic strategy for many hematologic diseases and genetic disorders. Therefore, a variety of ex vivo expansion techniques have been developed, however these systems were not well done to get long term HSCs (LT-HSCs) which have a long term hematopoietic reconstitution ability. As the reasons, it is considered that the factors associating with the proliferation and self-renewal of LT-HSCs have not been clear yet. To obtain the factors to stimulate the proliferation and self-renewal of LT-HSCs, various conditioned media were evaluated. The supernatants of COS-1 cells transfected with cDNA cording for RelA (one of nuclear factor kappa B subunits) stimulated the proliferation of human CD34+ cells derived from umbilical cord blood (UCB) and increased the number of CFU-Mix strongest of all evaluated conditioned media. 60 liters of the supernatants of COS-1 cells transfected RelA genes were separated by column chromatography purifications. LC-MS/MS analysis of the final active fraction provided the information of hepatoma-derived growth factor (HDGF) as a growth factor. HDGF is a 24kD heparin-binding protein and has reported to stimulate the proliferation in various types of cells including fibroblasts, endothelial cells and hepatoma cells, its receptor(s) and signaling remain unclear, moreover, has no known function in hematopoiesis. The recombinant human HDGF indicated the ability to enhance the proliferation of CD34+ cells dose-dependently and increased the number of CFU-Mix in combination with cytokines compared to cytokines alone, especially HDGF showed the strongest synergy effect in a combination with TPO in all combinations of cytokines. Next, uncultured (UC) CD34+ cells, the cells of an equal initial number of CD34+ cells after the serum-free condition cultures in the presence of TPO alone (T), HDGF alone (H) and HDGF+TPO (HT) were transplanted into sublethally irradiated NOG (NOD/Shi-scid,IL-2RγKO) mice. HT increased the number of CD34+CD38- cells compared to UC, T and H. Analysis of CD34+CD38- cells in bone marrow cells of NOG mice 24 weeks after transplantation revealed that the mean of absolute number of CD34+CD38- cells in HT group showed about 4-fold, that in H group showed about 3-fold compared to that in UC group, however, that in T group were not detected.These results indicated that HT increased HSCs including short term and long term HSCs. In order to investigate whether HDGF could increase the number of LT-HSCs, serial transplantation experiment was carried out. Uncultured CD34+ cells and the CD34+ cells cultured with HT were transplanted into sublethally irradiated NOG mice. At 24 weeks after transplantation, the mean of absolute number of CD34+CD38- cells in HT group showed 6-fold compared to that in UC group, a half of total number of bone marrow cells from each mouse in both groups were transplanted into one secondary sublethally irradiated NOG mouse. Analysis of human hematopoietic cells in both group 20 weeks after transplantation revealed that multi-lineage human hematopoietic cells, such as CD3+ cells, CD19+ cells, CD33+ cells, CD235a+ cells, erythrocytes and platelets, were detected in all mice in HT group, but were not detected in all mice in UC group. The mean of absolute number of CD34+CD38- cells in bone marrow of HT group showed 30-fold compared to that of UC group. These results indicated that HDGF could increase the number of LT-HSCs. We showed here that the CD34+ cells cultured with HDGF can be transplanted to secondary hosts to give rise to long-term multilineage repopulation. Thus, HDGF is a novel factor to promote the proliferation of HSCs and plays an important role in hematopoiesis. HDGF will contribute the new HSCs expansion system development by using UCB for hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Ex Vivo Expansion of CD34+ Cells From Cord Blood, Bone Marrow, and Mobilized Peripheral Blood Using NANEX Nanofiber Scaffold

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4816-4816
Author(s):  
Stephen L Fischer ◽  
Jacqueline M Fonseca ◽  
Yukang Zhao ◽  
Linda L. Kelley ◽  
Ramasamy Sakthivel
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cell Phenotype ◽  
Cd34 Cells ◽  
Ex Vivo Culture ◽  
Mobilized Peripheral Blood ◽  
Stem Cell Phenotype

Abstract Abstract 4816 Hematopoietic stem cell (HSC) transplantation has become the standard of care for patients with hematologic cancers, anemia, and a variety of other malignant and non-malignant disorders, with greater than 50,000 such procedures being performed globally each year, according to the Worldwide Network for Blood and Marrow Transplantation. Although mobilized peripheral blood (MPB) has become a preferred source of HSCs for transplants, bone marrow (BM) and umbilical cord blood (UCB) are also frequently utilized. Regardless of source, several groups have reported that grafts containing lower total nucleated cell (TNC) and CD34+ cell doses contribute to delayed engraftment and higher graft failure rate. Therefore, methods to increase the total cell number while maintaining the progenitor phenotype, especially the CD34+ progenitor cells, from individual grafts would have a significant clinical impact. Ex vivo expansion of HSCs prior to transplantation is one approach that offers tremendous promise for increasing cell doses and improving clinical outcomes. In many ex vivo culture systems, HSCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSCs with a substrate. In the natural bone marrow microenvironment, HSCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D NANEX nanofiber based cell culture substrate. The functionalized NANEX substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSC proliferation while maintain the progenitors stem cell phenotype. Here, we present our recent work with the NANEX platform towards comparing and achieving a high yield ex vivo expansion of CD34+ cells from MPB, BM, and UCB. Additionally, through the use of flow cytometry and CFU assays, we quantify and characterize NANEX-expanded cells from each source. Furthermore, we compared NANEX to a variety of commercially available products and demonstrate that NANEX significantly improves expansion and reduces phenotype loss during ex vivo culture. Our data indicates that NANEX technology provides a robust ex vivo expansion of HSCs and, with further GMP and clinical development, offers great potential for clinical applications. Disclosures: No relevant conflicts of interest to declare.

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