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Extracellular matrix free microcarrier cultures of human pluripotent stem cells induced by inhibition of rock-myosin II signaling
19th Annual ISCT Meeting
ABSTRACTS
Oral Abstracts 1 DATA IN SUPPORT OF THE CLINICAL USE OF ADIPOSE DERIVED MSC: GROWTH, STORAGE, FUNCTION AND SAFETY AB Dietz, DJ Padley, GW Butler, JM Anderson, MG Sarr, AJ Windebank, SC Textor, YC Kudva, E Galanis, K Peng, DA Gastineau Mayo Clinic, Rochester, MN Mesenchymal stromal cells (MSC) are a promising treatment for autoimmune disorders and tissue regeneration. Our laboratory has focused on an autologous approach using adipose tissue (AT) derived MSC (adMSC). In an effort to move these therapeutic cells into the clinic, we have performed studies to optimize and characterize the processes and the cells as the foundation of the manufacturing protocols for our clinical trials. Early on, we developed a media supplement derived from human platelets that is the basis of our culture protocol (PLTMax, Mill Creek Lifesciences, Rochester, MN). We have used this protocol to grow adMSC from otherwise healthy patients undergoing bariatric surgeries and from >30 patients with a variety of diseases including ALS, Crohn’s disease, type 1 diabetes, and ovarian cancer. We saw no differences in growth kinetics or phenotype associated with underlying disease. We have successfully cultured adMSC to more than 25 population doublings without loss of growth rate or change in phenotype. This protocol typically yielded 1 109 MSC in 3 weeks from a starting product of 1-2 gms of AT. When thawed, cells frozen for more than a year retained their pre-freeze proliferation rate. adMSC inhibited dendritic cell maturation as well as inhibited dendritic cell mediated stimulation of CD4 T cells. Safety studies in rabbits, pigs and mice have shown that cells have expected bio-distribution. No malignant transformation was seen during the injection of more than one billion adMSC into immune deficient mice. These and other preliminary data have led to the opening of phase I clinical trials using these cells to treat ALS, multiple system atrophy, and renal stenosis. Two other applications are under review. 2 A TISSUE ENGINEERING CONSTRUCT COMPRISING HUMAN ENDOMETRIAL MESENCHYMAL STROMAL CELLS AND POLYAMIDE/GELATIN MESH EVALUATED IN A PRECLINICAL ANIMAL MODEL OF PELVIC ORGAN PROLAPSE REPAIR D Ulrich1, SL Edwards2, JF White2, C Su2, K Tan1, A Rosamilia1, JA Ramshaw2, JA Werkmeister2, CE Gargett1 1 Monash University, Clayton, Victoria, Australia, 2CSIRO, Clayton, Victoria, Australia Pelvic organ prolapse (POP) is the herniation of bladder, bowel and/or uterus into the vagina causing urinary incontinence and sexual dysfunction. POP results from childbirth injury. 11-19% of women will be treated for POP by reconstructive surgery with or without polypropylene mesh. Complication rates approach 30%. Our aim was to use a tissue engineering approach to determine whether human endometrial stromal cells (eMSC) purified from the regenerative uterine lining improves the performance of a composite polyamide/gelatin mesh in a rat model of wound repair. eMSC were isolated from hysterectomy tissue using W5C5-labelled magnetic beads, cultured to P6 and labelled with VybrantÒ DiO. Warp-knitted polyamide monofilament meshes were coated with 12% cross-linked gelatin, gamma-sterilized and coated with fibronectin. eMSC (250,000 cells) were seeded onto 25 10 mm meshes and implanted subcutaneously in the dorsum of immunocompromised nude rats for 7, 30, 60, 90 days (n ¼ 8/group). Controls were meshes without cells. Explanted samples were analysed by flow cytometry to detect DiO-labelled cells, by immunohistochemistry to assess foreign body reaction and tissue integration. Collagen III/I ratios were quantified by chemical assays, collagen organisation by birefringence and tensile testing by Instron. Implanted meshes were well tolerated. eMSC were detected in explants until 14 days post-implant. eMSC/meshes contained significantly fewer CD45+ leukocytes, CD68+ macrophages at 90 days, significantly increased M1/M2 macrophage ratio and neovascularisation at 7 days than controls (all P < 0.05). New collagen was observed in meshes with and without eMSC. By 90 days, there were improved biomechanical properties of eMSC/mesh, increased collagen type III/I ratios and more collagen organisation than controls.
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This tissue engineering approach, using eMSC delivered on polyamide/ gelatin composite mesh, reduced inflammatory cells around implanted mesh fibres, promoted neovascularisation and improved mesh distensibilty over time, suggesting that this might be an alternative novel approach for future treatment of POP. 3 INTRA ARTICULAR INJECTION OF AUTOLOGOUS BONE MARROW-DERIVED MESENCHYMAL STROMAL CELLS IN PATIENTS WITH MODERATE TO SEVERE OSTEOARTHRITIS SP Chin1,2, NN Wazir3, CY Cheok4, CY Wong5, KY Then6, SK Cheong7 1 Mawar Hospital, Negeri Sembilan, Malaysia, 2Beverly Wilshire Medical Centre, Kuala Lumpur, Malaysia, 3International Medical University, Negeri Sembilan, Malaysia, 4Penang Adventist Hospital, Penang, Malaysia, 5 Cytopeutics, Selangor, Malaysia, 6Cryocord, Selangor, Malaysia, 7Tunku Abdul Rahman University, Selangor, Malaysia Background: Bone marrow-derived mesenchymal stromal cells (BMMSC) can be expanded ex vivo which have the ability to regenerate cartilage for accelerated healing of the knee as demonstrated by animal studies and early clinical reports. In this study we have evaluated the safety and feasibility of using autologous BMMSC as an intra-articular injection for the treatment of symptomatic moderate to severe osteoarthritis. Methods: Fifteen patients with symptomatic moderate to severe knee osteoarthritis were recruited. All patients have persistent non-improving or deteriorating pain despite regular oral analgesics and multiple hyaluronic injections. Autologous BMMSC was resuspended in a mixture of hyaluronic acid and autologous platelet rich plasma before intra-articular injection procedure. Patients were assessed and followed-up using the Oxford Knee Score (OKS) and magnetic resonance imaging (MRI) for up to 12 months. Results: The mean OKS at 6 and 12 months after BMMSC injection increased significantly (42.6 6.2 and 44.8 8.1) when compared to baseline scores (35.2 6.5). At 12 months, an improvement of OKS of 4 points or more was observed in 10 patients when compare to their baseline scores for both knees while 2 patients experienced improvement in the right knee only. MRI at 12 months post-BMMSC treatment showed noticeable improvement in 60% of patients including mean increase in cartilage thickness from baseline, resolution of subchondral cysts and reduction of effusion. Conclusion: Autologous BMMSC injection is safe, feasible and may be beneficial for the symptomatic treatment of patients with moderate to severe osteoarthritis who have failed conventional treatment. 4 EXTRACELLULAR MATRIX FREE MICROCARRIER CULTURES OF HUMAN PLURIPOTENT STEM CELLS INDUCED BY INHIBITION OF ROCK-MYOSIN II SIGNALING A Chen, Y Lim, X Chen, S Reuveny, SK Oh Bioprocessing Technology Institute, Singapore, Singapore Large quantities of human pluripotent stem cells (hPSC) needed for therapeutic applications can be obtained in scalable suspended microcarrier cultures. However, these microcarriers have to coated with animal or human extracellular matrix (ECM) proteins which can present safety risks, and/or are very expensive for large scale use. This study demonstrates that human embryonic stem cells (HES-3, H7) and induced pluripotent stem cell (IMR90) can be propagated on non-coated positively charged cellulose microcarriers in serum free medium containing ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. Dephosphorylation of myosin phosphotase 1 (MYPT1) and myosin light chain 2 (MLC2) were observed in the presence of these two inhibitors suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM-coating free microcarriers. Cells were propagated on the non-coated microcarriers for at least 15 passages while maintaining pluripotency and karyotype stability. Scalability of this platform was demonstrated in 100 ml spinner flask resulting in cell yields of 2.3 106 cells/ml (HES-3) after 5 days of growth. The capability of these cells to differentiate into the three primary lineages was demonstrated in in-vitro embryoid bodies and in-vivo teratoma formation studies. Moreover, directed differentiation to PSA-NCAM+ neural progenitor cells was demonstrated, high cell yields (9.1 1.2 106 cells/ml) and expression levels (91 1.1% cells expressing polysialylated neuronal cell adhesion molecule (PSA-NCAM)) were obtained. This defined serum- and coating- free scalable microcarrier culturing system
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Oral abstracts
can serve as a safe and less expensive method for generating large amounts of human pluripotent stem cells for cell therapies. 5 HUMAN EMBRYONIC STEM CELLS EXHIBIT ALTRUISTIC BEHAVIOR IN THE MICROENVIRONMENT OF OXIDATIVE STRESS B Das1, R Tsuchida2, R Bayat-Mokhtari3, DW Felsher1, H Yeger4 1 Stanford University, Stanford, CA, 2Tokyo Dental University, Tokyo, Japan, 3 Hospital for Sick Children, Toronto, ON, Canada, 4Hospital for Sick Children, Toroto, ON, Canada Altruism is a cytoprotective mechanism having deep evolutionary root. It implies a defense mechanism of cytoprotection, where a few cells sacrifice their own fitness to increase the fitness of the population. We considered the potential altruistic behavior of stem cells when exposed to extreme microenvironment prevalent in the area of injury/inflammation. We considered this possibility based on several clinical reports that although stem cells including human embryonic stem cells (hESCs) exert cytoprotective activity in the area of tissue injury, the engrafting of viable donor stem cells at the site of injury is very minimal. This is expected, because, hypoxia/oxidative stress prevailing in the area of injury could activate p53, leading to death and differentiation of donor stem cells. Therefore, stem cells might have evolved cytoprotective mechanism without engrafting. Using the hESCs as a model, we indeed discovered an altruistic mechanism that allow a small subset of hESCs to protect the rest of the population from DNA-damage, differentiation and death in the microenvironment of oxidative stress. We found that within the hESCs, the SSEA3+/ ABCG2+ fraction undergoes a transient state of reprogramming to a low p53 and high HIF-2a state of transcriptional activity in the microenvironment of extreme hypoxia (<0.01% Oxygen)/re-oxygenation. This state can be sustained for a period of two-weeks and is associated with enhanced transcriptional activity of Oct-4 and Nanog, and high secretion of glutathione. Conditioned medium (CM) obtained from the post-hypoxia SSEA3+/ABCG2+ hESCs showed cytoprotection of SSEA3+/ABCG2- cells from oxidative-stressinduced p53 activation. siRNA inhibition of ABCG2 reduced glutathione in the CM, and also diminished the cytoprotective activity. We then demonstrated that the underlying molecular mechanism of this transient phenotype of “enhanced stemness” involved an altered state of the p53/MDM2 oscillation system. Our study indicates the potential presence of an altruistic mechanism of stem cell cytoprotection.
cell gene expression and suppressed the genes activated during effector T cell differentiation. BIO promoted Tn cell divisions induced by IL7 suggesting that GSK-3b inhibition elicits Tn cell expansion promoting crosstalk between Wnt and IL7/IL7Rs signalling. We propose that GSK-3b inhibition can potentially promote T-cell responses in the recipients of stem cell transplant, particularly in adult patients with impaired thymic function. 7 BONE MARROW VERSUS PBPC FOR ALLOGENEIC STEM CELL TRANSPLANT: DEFINING THE OPTIMAL GRAFT I McNiece1, S Sivajothoi1, E Shpall1, N Kenyon2, M Korbling1, C Hosing1 1 The University of Texas MD Anderson Cancer Center, Houston, TX, 2The University of Miami, Miami, FL Randomized trials comparing bone marrow (BM) to G-CSF mobilized peripheral blood progenitor cell (G-PBPC) products for unrelated-donor transplantation, resulted in no significant difference in survival. G-PBPCs were associated with faster engraftment and reduced risk of graft failure but also a significant increase in the risk of chronic graft versus host disease (cGVHD). This has resulted in the proposal that bone marrow be used in preference to G-PBPC for the majority of unrelated-donor transplants. BM grafts contain two types of stem cells: hematopoietic (HSCs) and mesenchymal (MSCs) stem cells. As MSCs have been shown to be immune suppressive and have been used to reverse GVHD, we evaluated whether G-PBPC contain MSCs as well as HSCs. MNCs were isolated from normal donor G-PBPCs and cultured for MSC in flasks in alpha MEM plus 20% FBS. MSCs failed to grow from G-PBPC and the products lacked CD271+CD45- cells. We therefore evaluated different mobilization agents and demonstrated in mice that AMD3100 could mobilize MSCs. Treatment of mice with G-CSF alone failed to mobilize MSCs, while the combination of G-CSF plus AMD3100 resulted in significant levels of MSCs in the peripheral blood of treated mice. These studies have been repeated in a non-human primate. Treatment with G-CSF for days 1 to 5 plus AMD3100 on day 5 resulted in significant levels of MSCs in the peripheral blood collected on day 6. Based upon these data we propose that mobilization of normal donors with G-CSF plus Mozobil will be effective in mobilizing both HSCs and MSCs and generate the optimal stem cell graft providing both rapid engraftment and cGVHD equivalent to BM. We have initiated a clinical trial at MD Anderson to evaluate the safety of G-CSF plus Mozobil mobilization in normal donors and will determine the potential to mobilize MSCs.
6 GSK-3b INHIBITION PROMOTES T CELL RECONSTITUTION AND PRESERVES NAÏVE T CELL PHENOTYPE IN MICE TRANSPLANTED WITH HUMAN HAEMATOPOIETIC STEM CELLS A Dolnikov, S Shen, G Klamer, N Xu, TA O’Brien Sydney Children’s Hospital, Level 3 Clinical Sciences Building High Street, Australia
8 LOW DOSE LENALIDOMIDE INDUCTION FOLLOWED BY AUTOLOGOUS TRANSPLANTATION IN UNTREATED PATIENTS WITH MYELOMA IS ASSOCIATED WITH ADEQUATE COLLECTION OF HAEMATOPOIETIC AND DENDRITIC CELL PRECURSORS AND HIGH RESPONSE RATES A Khot, R Sedunary, K Stokes, M Loudovaris, D Wall, M Prince, D Ritchie, S Harrison Peter MacCallum Cancer Centre, East Melbourne, Australia
Stem cell transplantation (SCT) has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Immunologic reconstitution following stem cell transplantation is a critical component for successful outcome. Chemotherapy and pre-conditioning impair thymic function leading to delayed T cell regeneration. The lack of T cells with a broad T-cell receptor repertoire leads to an increased risk for opportunistic infections and leukaemia relapse. Therefore, enhancing immune reconstitution is important. The Wingless pathway (Wnt) was identified as an important regulator of T cell function. In normally functioning thymus thymic epithelial cells provide Wnt signals to developing thymocytes. Chemotherapy and pre-conditioning lead to Wnt depletion due to impaired thymic function. Therefore, providing right dosage of Wnt to the thymocytes following preconditioning may provide specific therapeutic option for SCT patients. Here we show that conditional Wnt activation using small molecule inhibitor of Glycogen Synthase Kinase 3b (GSK-3b), 6-bromoindirubin 30 -oxime (BIO), promotes human T cell reconstitution in immuno-compromised mice transplanted with cord blood CD34+ stem cells. BIO treatment did not modulate the proportion of early lymphoid CD34+CD7+ progenitor cells in the bone marrow and thymus but promoted T cell expansion in periphery and increased naïve T (Tn) cell pool preventing effector differentiation during homeostatic T cell proliferation. GSK-3b inhibition in mature human T cells preserved Tn
Lenalidomide 25 mg D1-21 with dexamethasone 40 mg weekly is effective in first line and maintenance treatment of myeloma. Higher doses of lenalidomide and dexamethasone are associated with increased toxicity. We examined efficacy of low dose lenalidomide and dexamethasone induction followed by consolidation haematopoietic stem cell Transplantation HSCT in LITVacc, a phase II study of Lenalidomide Induction, HSCT and adjuvant Vaccination with autologous dendritic cells (DC) loaded with autologous tumour cell lysate (ATCL) and lenalidomide maintenance. Subjects: Newly diagnosed HSCT eligible myeloma patients. Induction: Lenalidomide 15 mg days 1-21 and dexamethasone 20 mg weekly, 428 day cycles. HSC mobilization: cyclophosphamide 2-4 gm/m2 and G-CSF 10 mcg/ kg/day. HSCT conditioning: melphalan 140-200 mg/m2. Maintenance: commenced D21-35 post HSCT: lenalidomide 25 mg D1-21 (minimum 1228 day cycles) DC vaccination (D1 C1-6) (n¼10). 20 patients have completed induction, HSCT and commenced maintenance. Median age 57.5 range (44-70). Median CD34+cells collected ¼ 10.8106 (4.9 - 40.6) over a median of 3 (2-6) days. Sufficient DC for six (1106 cell) vaccinations, meeting release criteria, were generated in 12 patients, with median DC yield¼165106 (45-298) and purity of 84.2% (72-98). Median number of CD34+cells infused was 4.8106 (2.9-11.5) with median time to recovery of neutrophils and platelets of 12 (9-18) and 11(8-25) days respectively. Post
ABSTRACTS
Oral Abstracts 1 DATA IN SUPPORT OF THE CLINICAL USE OF ADIPOSE DERIVED MSC: GROWTH, STORAGE, FUNCTION AND SAFETY AB Dietz, DJ Padley, GW Butler, JM Anderson, MG Sarr, AJ Windebank, SC Textor, YC Kudva, E Galanis, K Peng, DA Gastineau Mayo Clinic, Rochester, MN Mesenchymal stromal cells (MSC) are a promising treatment for autoimmune disorders and tissue regeneration. Our laboratory has focused on an autologous approach using adipose tissue (AT) derived MSC (adMSC). In an effort to move these therapeutic cells into the clinic, we have performed studies to optimize and characterize the processes and the cells as the foundation of the manufacturing protocols for our clinical trials. Early on, we developed a media supplement derived from human platelets that is the basis of our culture protocol (PLTMax, Mill Creek Lifesciences, Rochester, MN). We have used this protocol to grow adMSC from otherwise healthy patients undergoing bariatric surgeries and from >30 patients with a variety of diseases including ALS, Crohn’s disease, type 1 diabetes, and ovarian cancer. We saw no differences in growth kinetics or phenotype associated with underlying disease. We have successfully cultured adMSC to more than 25 population doublings without loss of growth rate or change in phenotype. This protocol typically yielded 1 109 MSC in 3 weeks from a starting product of 1-2 gms of AT. When thawed, cells frozen for more than a year retained their pre-freeze proliferation rate. adMSC inhibited dendritic cell maturation as well as inhibited dendritic cell mediated stimulation of CD4 T cells. Safety studies in rabbits, pigs and mice have shown that cells have expected bio-distribution. No malignant transformation was seen during the injection of more than one billion adMSC into immune deficient mice. These and other preliminary data have led to the opening of phase I clinical trials using these cells to treat ALS, multiple system atrophy, and renal stenosis. Two other applications are under review. 2 A TISSUE ENGINEERING CONSTRUCT COMPRISING HUMAN ENDOMETRIAL MESENCHYMAL STROMAL CELLS AND POLYAMIDE/GELATIN MESH EVALUATED IN A PRECLINICAL ANIMAL MODEL OF PELVIC ORGAN PROLAPSE REPAIR D Ulrich1, SL Edwards2, JF White2, C Su2, K Tan1, A Rosamilia1, JA Ramshaw2, JA Werkmeister2, CE Gargett1 1 Monash University, Clayton, Victoria, Australia, 2CSIRO, Clayton, Victoria, Australia Pelvic organ prolapse (POP) is the herniation of bladder, bowel and/or uterus into the vagina causing urinary incontinence and sexual dysfunction. POP results from childbirth injury. 11-19% of women will be treated for POP by reconstructive surgery with or without polypropylene mesh. Complication rates approach 30%. Our aim was to use a tissue engineering approach to determine whether human endometrial stromal cells (eMSC) purified from the regenerative uterine lining improves the performance of a composite polyamide/gelatin mesh in a rat model of wound repair. eMSC were isolated from hysterectomy tissue using W5C5-labelled magnetic beads, cultured to P6 and labelled with VybrantÒ DiO. Warp-knitted polyamide monofilament meshes were coated with 12% cross-linked gelatin, gamma-sterilized and coated with fibronectin. eMSC (250,000 cells) were seeded onto 25 10 mm meshes and implanted subcutaneously in the dorsum of immunocompromised nude rats for 7, 30, 60, 90 days (n ¼ 8/group). Controls were meshes without cells. Explanted samples were analysed by flow cytometry to detect DiO-labelled cells, by immunohistochemistry to assess foreign body reaction and tissue integration. Collagen III/I ratios were quantified by chemical assays, collagen organisation by birefringence and tensile testing by Instron. Implanted meshes were well tolerated. eMSC were detected in explants until 14 days post-implant. eMSC/meshes contained significantly fewer CD45+ leukocytes, CD68+ macrophages at 90 days, significantly increased M1/M2 macrophage ratio and neovascularisation at 7 days than controls (all P < 0.05). New collagen was observed in meshes with and without eMSC. By 90 days, there were improved biomechanical properties of eMSC/mesh, increased collagen type III/I ratios and more collagen organisation than controls.
S5
This tissue engineering approach, using eMSC delivered on polyamide/ gelatin composite mesh, reduced inflammatory cells around implanted mesh fibres, promoted neovascularisation and improved mesh distensibilty over time, suggesting that this might be an alternative novel approach for future treatment of POP. 3 INTRA ARTICULAR INJECTION OF AUTOLOGOUS BONE MARROW-DERIVED MESENCHYMAL STROMAL CELLS IN PATIENTS WITH MODERATE TO SEVERE OSTEOARTHRITIS SP Chin1,2, NN Wazir3, CY Cheok4, CY Wong5, KY Then6, SK Cheong7 1 Mawar Hospital, Negeri Sembilan, Malaysia, 2Beverly Wilshire Medical Centre, Kuala Lumpur, Malaysia, 3International Medical University, Negeri Sembilan, Malaysia, 4Penang Adventist Hospital, Penang, Malaysia, 5 Cytopeutics, Selangor, Malaysia, 6Cryocord, Selangor, Malaysia, 7Tunku Abdul Rahman University, Selangor, Malaysia Background: Bone marrow-derived mesenchymal stromal cells (BMMSC) can be expanded ex vivo which have the ability to regenerate cartilage for accelerated healing of the knee as demonstrated by animal studies and early clinical reports. In this study we have evaluated the safety and feasibility of using autologous BMMSC as an intra-articular injection for the treatment of symptomatic moderate to severe osteoarthritis. Methods: Fifteen patients with symptomatic moderate to severe knee osteoarthritis were recruited. All patients have persistent non-improving or deteriorating pain despite regular oral analgesics and multiple hyaluronic injections. Autologous BMMSC was resuspended in a mixture of hyaluronic acid and autologous platelet rich plasma before intra-articular injection procedure. Patients were assessed and followed-up using the Oxford Knee Score (OKS) and magnetic resonance imaging (MRI) for up to 12 months. Results: The mean OKS at 6 and 12 months after BMMSC injection increased significantly (42.6 6.2 and 44.8 8.1) when compared to baseline scores (35.2 6.5). At 12 months, an improvement of OKS of 4 points or more was observed in 10 patients when compare to their baseline scores for both knees while 2 patients experienced improvement in the right knee only. MRI at 12 months post-BMMSC treatment showed noticeable improvement in 60% of patients including mean increase in cartilage thickness from baseline, resolution of subchondral cysts and reduction of effusion. Conclusion: Autologous BMMSC injection is safe, feasible and may be beneficial for the symptomatic treatment of patients with moderate to severe osteoarthritis who have failed conventional treatment. 4 EXTRACELLULAR MATRIX FREE MICROCARRIER CULTURES OF HUMAN PLURIPOTENT STEM CELLS INDUCED BY INHIBITION OF ROCK-MYOSIN II SIGNALING A Chen, Y Lim, X Chen, S Reuveny, SK Oh Bioprocessing Technology Institute, Singapore, Singapore Large quantities of human pluripotent stem cells (hPSC) needed for therapeutic applications can be obtained in scalable suspended microcarrier cultures. However, these microcarriers have to coated with animal or human extracellular matrix (ECM) proteins which can present safety risks, and/or are very expensive for large scale use. This study demonstrates that human embryonic stem cells (HES-3, H7) and induced pluripotent stem cell (IMR90) can be propagated on non-coated positively charged cellulose microcarriers in serum free medium containing ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. Dephosphorylation of myosin phosphotase 1 (MYPT1) and myosin light chain 2 (MLC2) were observed in the presence of these two inhibitors suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM-coating free microcarriers. Cells were propagated on the non-coated microcarriers for at least 15 passages while maintaining pluripotency and karyotype stability. Scalability of this platform was demonstrated in 100 ml spinner flask resulting in cell yields of 2.3 106 cells/ml (HES-3) after 5 days of growth. The capability of these cells to differentiate into the three primary lineages was demonstrated in in-vitro embryoid bodies and in-vivo teratoma formation studies. Moreover, directed differentiation to PSA-NCAM+ neural progenitor cells was demonstrated, high cell yields (9.1 1.2 106 cells/ml) and expression levels (91 1.1% cells expressing polysialylated neuronal cell adhesion molecule (PSA-NCAM)) were obtained. This defined serum- and coating- free scalable microcarrier culturing system
S6
Oral abstracts
can serve as a safe and less expensive method for generating large amounts of human pluripotent stem cells for cell therapies. 5 HUMAN EMBRYONIC STEM CELLS EXHIBIT ALTRUISTIC BEHAVIOR IN THE MICROENVIRONMENT OF OXIDATIVE STRESS B Das1, R Tsuchida2, R Bayat-Mokhtari3, DW Felsher1, H Yeger4 1 Stanford University, Stanford, CA, 2Tokyo Dental University, Tokyo, Japan, 3 Hospital for Sick Children, Toronto, ON, Canada, 4Hospital for Sick Children, Toroto, ON, Canada Altruism is a cytoprotective mechanism having deep evolutionary root. It implies a defense mechanism of cytoprotection, where a few cells sacrifice their own fitness to increase the fitness of the population. We considered the potential altruistic behavior of stem cells when exposed to extreme microenvironment prevalent in the area of injury/inflammation. We considered this possibility based on several clinical reports that although stem cells including human embryonic stem cells (hESCs) exert cytoprotective activity in the area of tissue injury, the engrafting of viable donor stem cells at the site of injury is very minimal. This is expected, because, hypoxia/oxidative stress prevailing in the area of injury could activate p53, leading to death and differentiation of donor stem cells. Therefore, stem cells might have evolved cytoprotective mechanism without engrafting. Using the hESCs as a model, we indeed discovered an altruistic mechanism that allow a small subset of hESCs to protect the rest of the population from DNA-damage, differentiation and death in the microenvironment of oxidative stress. We found that within the hESCs, the SSEA3+/ ABCG2+ fraction undergoes a transient state of reprogramming to a low p53 and high HIF-2a state of transcriptional activity in the microenvironment of extreme hypoxia (<0.01% Oxygen)/re-oxygenation. This state can be sustained for a period of two-weeks and is associated with enhanced transcriptional activity of Oct-4 and Nanog, and high secretion of glutathione. Conditioned medium (CM) obtained from the post-hypoxia SSEA3+/ABCG2+ hESCs showed cytoprotection of SSEA3+/ABCG2- cells from oxidative-stressinduced p53 activation. siRNA inhibition of ABCG2 reduced glutathione in the CM, and also diminished the cytoprotective activity. We then demonstrated that the underlying molecular mechanism of this transient phenotype of “enhanced stemness” involved an altered state of the p53/MDM2 oscillation system. Our study indicates the potential presence of an altruistic mechanism of stem cell cytoprotection.
cell gene expression and suppressed the genes activated during effector T cell differentiation. BIO promoted Tn cell divisions induced by IL7 suggesting that GSK-3b inhibition elicits Tn cell expansion promoting crosstalk between Wnt and IL7/IL7Rs signalling. We propose that GSK-3b inhibition can potentially promote T-cell responses in the recipients of stem cell transplant, particularly in adult patients with impaired thymic function. 7 BONE MARROW VERSUS PBPC FOR ALLOGENEIC STEM CELL TRANSPLANT: DEFINING THE OPTIMAL GRAFT I McNiece1, S Sivajothoi1, E Shpall1, N Kenyon2, M Korbling1, C Hosing1 1 The University of Texas MD Anderson Cancer Center, Houston, TX, 2The University of Miami, Miami, FL Randomized trials comparing bone marrow (BM) to G-CSF mobilized peripheral blood progenitor cell (G-PBPC) products for unrelated-donor transplantation, resulted in no significant difference in survival. G-PBPCs were associated with faster engraftment and reduced risk of graft failure but also a significant increase in the risk of chronic graft versus host disease (cGVHD). This has resulted in the proposal that bone marrow be used in preference to G-PBPC for the majority of unrelated-donor transplants. BM grafts contain two types of stem cells: hematopoietic (HSCs) and mesenchymal (MSCs) stem cells. As MSCs have been shown to be immune suppressive and have been used to reverse GVHD, we evaluated whether G-PBPC contain MSCs as well as HSCs. MNCs were isolated from normal donor G-PBPCs and cultured for MSC in flasks in alpha MEM plus 20% FBS. MSCs failed to grow from G-PBPC and the products lacked CD271+CD45- cells. We therefore evaluated different mobilization agents and demonstrated in mice that AMD3100 could mobilize MSCs. Treatment of mice with G-CSF alone failed to mobilize MSCs, while the combination of G-CSF plus AMD3100 resulted in significant levels of MSCs in the peripheral blood of treated mice. These studies have been repeated in a non-human primate. Treatment with G-CSF for days 1 to 5 plus AMD3100 on day 5 resulted in significant levels of MSCs in the peripheral blood collected on day 6. Based upon these data we propose that mobilization of normal donors with G-CSF plus Mozobil will be effective in mobilizing both HSCs and MSCs and generate the optimal stem cell graft providing both rapid engraftment and cGVHD equivalent to BM. We have initiated a clinical trial at MD Anderson to evaluate the safety of G-CSF plus Mozobil mobilization in normal donors and will determine the potential to mobilize MSCs.
6 GSK-3b INHIBITION PROMOTES T CELL RECONSTITUTION AND PRESERVES NAÏVE T CELL PHENOTYPE IN MICE TRANSPLANTED WITH HUMAN HAEMATOPOIETIC STEM CELLS A Dolnikov, S Shen, G Klamer, N Xu, TA O’Brien Sydney Children’s Hospital, Level 3 Clinical Sciences Building High Street, Australia
8 LOW DOSE LENALIDOMIDE INDUCTION FOLLOWED BY AUTOLOGOUS TRANSPLANTATION IN UNTREATED PATIENTS WITH MYELOMA IS ASSOCIATED WITH ADEQUATE COLLECTION OF HAEMATOPOIETIC AND DENDRITIC CELL PRECURSORS AND HIGH RESPONSE RATES A Khot, R Sedunary, K Stokes, M Loudovaris, D Wall, M Prince, D Ritchie, S Harrison Peter MacCallum Cancer Centre, East Melbourne, Australia
Stem cell transplantation (SCT) has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Immunologic reconstitution following stem cell transplantation is a critical component for successful outcome. Chemotherapy and pre-conditioning impair thymic function leading to delayed T cell regeneration. The lack of T cells with a broad T-cell receptor repertoire leads to an increased risk for opportunistic infections and leukaemia relapse. Therefore, enhancing immune reconstitution is important. The Wingless pathway (Wnt) was identified as an important regulator of T cell function. In normally functioning thymus thymic epithelial cells provide Wnt signals to developing thymocytes. Chemotherapy and pre-conditioning lead to Wnt depletion due to impaired thymic function. Therefore, providing right dosage of Wnt to the thymocytes following preconditioning may provide specific therapeutic option for SCT patients. Here we show that conditional Wnt activation using small molecule inhibitor of Glycogen Synthase Kinase 3b (GSK-3b), 6-bromoindirubin 30 -oxime (BIO), promotes human T cell reconstitution in immuno-compromised mice transplanted with cord blood CD34+ stem cells. BIO treatment did not modulate the proportion of early lymphoid CD34+CD7+ progenitor cells in the bone marrow and thymus but promoted T cell expansion in periphery and increased naïve T (Tn) cell pool preventing effector differentiation during homeostatic T cell proliferation. GSK-3b inhibition in mature human T cells preserved Tn
Lenalidomide 25 mg D1-21 with dexamethasone 40 mg weekly is effective in first line and maintenance treatment of myeloma. Higher doses of lenalidomide and dexamethasone are associated with increased toxicity. We examined efficacy of low dose lenalidomide and dexamethasone induction followed by consolidation haematopoietic stem cell Transplantation HSCT in LITVacc, a phase II study of Lenalidomide Induction, HSCT and adjuvant Vaccination with autologous dendritic cells (DC) loaded with autologous tumour cell lysate (ATCL) and lenalidomide maintenance. Subjects: Newly diagnosed HSCT eligible myeloma patients. Induction: Lenalidomide 15 mg days 1-21 and dexamethasone 20 mg weekly, 428 day cycles. HSC mobilization: cyclophosphamide 2-4 gm/m2 and G-CSF 10 mcg/ kg/day. HSCT conditioning: melphalan 140-200 mg/m2. Maintenance: commenced D21-35 post HSCT: lenalidomide 25 mg D1-21 (minimum 1228 day cycles) DC vaccination (D1 C1-6) (n¼10). 20 patients have completed induction, HSCT and commenced maintenance. Median age 57.5 range (44-70). Median CD34+cells collected ¼ 10.8106 (4.9 - 40.6) over a median of 3 (2-6) days. Sufficient DC for six (1106 cell) vaccinations, meeting release criteria, were generated in 12 patients, with median DC yield¼165106 (45-298) and purity of 84.2% (72-98). Median number of CD34+cells infused was 4.8106 (2.9-11.5) with median time to recovery of neutrophils and platelets of 12 (9-18) and 11(8-25) days respectively. Post