- Email: [email protected]
Optimization of xeno-free expansion conditions for human umbilical cord-derived perivascular cells using functional immunophenotyping and in vitro potency assays
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Poster Abstracts
control strategy developed. MSC identity and quality characteristics have been assessed following our novel harvesting and cryopreservation process with MSCs demonstrating full ISCT criteria as well as maintaining monolayer outgrowth and colony-forming efficiency. Increased inter-donor consistency of MSC product has been demonstrated in long term serum-free culture in terms of growth, metabolomics, colony-forming efficiency, osteogenic potential and expression of key genes involved in immunomodulatory and angiogenic function. These findings have implications in the development of both autologous and allogeneic cell therapy bioprocesses, with consistency gains having great potential to provide significant cost savings in process development and validation. This convergence in product yield and quality has been driven by the systematic development of a controlled, scalable and serum-free MSC expansion, harvest and cryopreservation process.
Figure 1. Driving consistency into the MSC expansion process. (A) increased consistency in MSC growth for serum-free media (SFM) compared to bovine-serum (FBS) and human-serum (HPL) between multiple donors & (B) increased consistency in MSC quality attributes in SFM between donors. 283 WILL NOT BE PRESENTED 284 IMPLEMENTATION OF ACADEMIC ADVANCED THERAPY MEDICINAL PRODUCTS IN CLINICAL PRACTICE IN THE NETHERLANDS Sd Wilde1, LA Veltrop-Duits1, M Hoozemans-Strik2, J Veenman3, H-J Guchelaar1, M Zandvliet1, P Meij1 1 Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, Netherlands, 2Dutch Cancer Society, Amsterdam, Netherlands, 3Netherlands Research Organisation of Health Research and Development (ZonMw), The Hague, Netherlands Advanced Therapy Medicinal Products (ATMPs), including gene therapy products (GTMPs), somatic cell therapy products (sCTMP) and tissue engineered products (TEP), can only be applied under the European regulatory framework, (1) within a clinical study protocol, (2) within the hospital exemption (HE) and (3) with an EMA (European Medicines Agency) Marketing Authorization. Until now only four ATMPs (ChondrocelectÒ, GlyberaÒ, MACIÒ and ProvengeÒ) succeeded to achieve EMA approval, despite the fact that a lot of ATMPs were tested in clinical trials. Most ATMPs developed in academia, which appear to be safe and effective, remain in developmental phase. This project aims to identify how academic ATMPs can efficiently become available for regular clinical care. The association between variables and success or failure in the route towards implementation in regular care will be analyzed. From different academia in The Netherlands 49 ATMPs** were selected. Variables that could have impact on either the development or the implementation of the products are analyzed using interviews with research groups and stakeholders. Financing is the main hurdle in ATMP development. The intention to register an ATMP is clear for the GTMP projects in contrast to most CTMP projects. Intellectual property (IP) has been secured in 52% and HE was used in 17% of the products with registration intention. In 64% of the ‘uncertain about registration group’ proof of efficacy is considered essential before thinking about further implementation in regular clinical care. The impossibility to further modify a product after registration is for research groups a motive to not registrate their product. Based on the results, optimal routes for academic development and implementation of ATMPs will be designed and communicated to the academic field.
** This project and the selected ATMPs are supported by Dutch Cancer Society (DCS) and Netherlands Research Organisation of Health Research and Development (ZonMw). 285 OPTIMIZED MANUFACTURING PROCESS FOR THE GENERATION OF CLINICAL GRADE CAR T CELLS P Bajgain1, R Mucharla1, N Watanabe1, J Wilson2, U Anurathapan1, N Lapteva1, H Heslop1, C Rooney1, M Brenner1, AM Leen1, J Vera1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, Houston Methodist Hospital, Houston, Texas, United States, 2Wilson Wolf Manufacturing, St Paul, Minnesota, United States Although adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has shown promising clinical responses, prolonged and complicated cell production process has limited its broader application. To simplify CAR T cell manufacturing, we assessed whether cell expansion could be improved by: (i) supplementing cultures with artificial antigen presenting cells (a-APC), and (ii) growing cells in a gas permeable culture device (G-Rex). As a proof of concept, CAR T cells targeting the prostate cancer antigen, PSCA. By retroviral transduction of K562 cells to express PSCA, we generated an a-APC cell line, which resulted in a 3.4 fold greater CAR T cell expansion relative to IL2 alone. Moreover, CAR-T cells were enriched in the a-APC cultures, increasing from 43.47.6% to 85.59.2% (n¼3) in 10 days, unlike IL2-expanded cells where they remained unchanged (43.37.6% to 43.44.1% on day 10). Although this a-APC-based approach induced selective CAR-T cell expansion, using traditional 24-well tissue culture-treated plates made the manufacturing process labor and time intensive. Thus, to simplify cell production, we assessed whether CAR-PSCA T cells with a-APC stimulation(1:2) could be expanded in a static GMP-compliant G-Rex 100M with a surface area of 100cm2. From an initial 25E+06 CAR T cells, we obtained 2963.8195.2E+06 cells (n¼3) in 10 days, a 118.6 fold increase using only 1 liter of media. As expected, the expanded population were enriched for transgenic CAR-T cells (43.47.6% - day 0 and 83.67.0% - day 10; n¼3). Importantly, T cells manufactured using this method were able to better retain anti-tumor activity and expression of memory and activation markers such as CD62L and CD25 when compared to cells maintained in conventional culture. In order to further simplify the manufacturing process, we have now developed a semi-automatic cell collection device that can be paired G-Rex 100M, which allows cell collection in a small volume (100ml) in under five minutes. 286 OPTIMIZATION OF XENO-FREE EXPANSION CONDITIONS FOR HUMAN UMBILICAL CORD-DERIVED PERIVASCULAR CELLS USING FUNCTIONAL IMMUNOPHENOTYPING AND IN VITRO POTENCY ASSAYS M Librach1, S Pereira1, L Maghen1, F Iqbal1,2, T Barretto1, K Park1, P Szaraz1,2, A Gauthier-Fisher1, C Librach1,3,2,4 1 CReATe Fertility Centre, Toronto, Ontario, Canada, 2Department of Physiology, University of Toronto, Toronto, Ontario, Canada, 3Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada, 4Department of Gynecology, Women’s College Hospital, Toronto, Ontario, Canada Xeno-free expansion of MSCs is important for their clinical translation to cell therapy. Recent reports demonstrated the maintenance or enhancement of proliferative potential, immunological and MSC properties in xeno-free culture conditions, yet very few studies assessed the expression of molecules and phenotypes that could impact the acute behavior of MSCs post-implantation. We isolated and expanded first trimester human umbilical cord-derived perivascular cells (FTM-PVCs) in 4 commercially availabe xeno-free human MSC culture media(A-D), as well as serum containing conditions(aMEM + 10% FBS). Media A did not support the expansion of FTM-PVC cultures. Only media B and aMEM + 10% FBS conditions supported the establishment of FTM-PVC cultures. We compared the expansive capacity and population doubling time (PDT) through 9 passages. With the exception of media C, which showed drastically reduced PDT at early passages, the other 3 zeno-free conditions were comparable to FBS-containing conditions. We assessed the immunophenotype of expanded FTM-PVCs at passages 3 and 6 using flow cytometry for surface markers that define MSCs. The majority of cells were CD90+ and CD44+ in all conditions. CD105 expression decreased in media C and D conditions. However, we observed that the expression of markers
21st ISCT Annual Meeting
associated with immunoprivilege(HLA-G), with acute responsiveness(CXCR4, CD49f, PDGFRbeta) or other cellular attributes(SSEA4, CD146, Oct4A, MMPs) varied dramatically between culture conditions, with media B giving expression levels closest to serum conditions for the majority of these markers. In vitro functional potency assays evaluated and compared the immunosuppressive, chemotaxis, extravasation and angiogenic properties of FTM-PVCs expanded in each condition. Our results suggest that while MSC properties are maintained in most xeno-free conditions, characteristics that could significantly impact acute behavior in vivo vary greatly between the culture conditions evaluated. 287 VALIDATION OF THE CliniMACS PRODIGY FOR MANUFACTURE OF TUMOR-PRIMED NATURAL KILLER CELLS (TpNK) H Bartley1, L Clarke2, M Lowdell1, ER Samuel1 1 Haematology, University College London, London, United Kingdom, 2Miltenyi Biotec, Bisley, United Kingdom The production of clinical-grade TpNK cells under good manufacturing practice (GMP) includes immunomagnetic isolation of NK (CD56+/CD3-) and NKT (CD56+/CD3+) cells, priming with tumor-lysate (TL) and removal of contaminating TL, QC testing and final release. Current methods result in CD56+ yields of w30% with purity of >95% but with significant TpNK loss following TL removal by density gradient separation in an open-system. This study aimed to validate the translation of TpNK manufacture onto the CliniMACS Prodigy to evaluate NK cell yield and purity and efficiency of TL removal within a closed-system. CD56+ NK cell isolation on the CliniMACS Prodigy was assessed in six donor leukocyte apheresates and TL removal experiments performed using the CTV-1 cell line as mock TpNK cells, co-cultured with TL in three validation runs. CD56+ enrichment and TL removal were performed using an enrichment program 2.0 and a lysate-ficol program respectively. CD56+ isolation resulted in a median purity of 92.3% (83.9-97.0%), yield of 66.5% (35.3-79.3%) and a median CD3+ T cell log depletion of 2.4 (2.0-2.5). Median viability was 96.7% (68.3-98.1%) and total processing time was 140 minutes. Assessment of TL removal by ficol-density gradient resulted in a median CTV-1 yield of 59.0% (48.0-68.0%), TL depletion of >90% and a median viability of 99.0% (95-99%), meeting acceptance criteria for GMP manufacture and TpNK cell final release. TpNK cell manufacture on the CliniMACS Prodigy is feasible, demonstrating improved CD56+ yields following isolation and improved recovery after TL removal. This will substantially reduce COGs and increase the TpNK cell dose from a single donor apheresis. Translation to a fully closed-process on the CliniMACS Prodigy will allow transfer of manufacturing into a Grade D GMP suite from a current Grade B laboratory achieving significant savings in GMP operating costs.
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288 PRENATAL AND POSTNATAL TRANSPLANTATION OF HUMAN AMNIOTIC FLUID STEM CELLS IN SPINAL MUSCULAR ATROPHY MICE S Shaw1,2, L-K Tsai3, P-J Cheng1 1 Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taoyuan, Taiwan, 2Institute for Women’s Health, University College London, London, United Kingdom, 3National Taiwan University, Taipei, Taiwan Human amniotic fluid is a promising resource of pluripotent stem cells and it makes fetal therapy using autologous transplantation possible. Human amniotic fluid stem (AFS) cells are a unique subgroup of human AF-derived stem cells that are isolated by C-Kit immunoselection, and which possess amazing capability of self-renewal and potential to differentiate. Several human stem cells have engrafted successfully in fetal mice via in-utero transplantations (IUT) that include the stem cells from fetal blood and bone marrow, but amniotic fluid yet. We designed IUT into spinal muscular atrophy (SMA) mice using human AFS cells. Adult SMA mice had been proved that could be treated with human AFS by other research group. We transplanted 100,000 human AFS cells per pup into the fetal mice (type I and type III SMA) intraperitoneally at E13 and 3 months postnatally. There were 89 fetuses from 9 dams received prenatal cell therapy with 62% survival rate. Three experimental groups were designed as human AFS cells, human fibroblasts or normal saline injection into fetuses for comparison. The preliminary results showed the engraft evidence in the PCR, flowcytomery and immunohistochemistry among multiple fetal organs especially in the skeletal muscles. However, we did not see the prolong survival for type I SMA (severe form) yet after in utero human AFS cells transplantation. The functional test of long-term survival in type III SMA showed significant improvement compared to untreated group with longer survival. This is the first study using human amniotic fluid stem cells as cell therapy source for prenatal transplantation into mice SMA model. For the clinical purpose, these cells either with mesenchymal or hematopoietic potential could be obtained prenatally for autologous cell fetal therapy, or storage the cells for postnatally tissue engendering in the future. 289 OVERCOMING THE TRANSLATIONAL CHALLENGES OF THE EFFECTIVE ADMINISTRATION AND DELIVERY OF CELLS A Lyness, N Medcalf, D Williams Wolfson School of Mechanical & Manufacturing Engineering, Loughborough University, Loughborough, Leicestershire, United Kingdom Of the few cell-based therapies widely available today the most mature are the treatment of blood-borne cancers and wound healing. The routes of administration for these applications are well understood and a great deal of research has resolved the many biological, pharmacological and engineering challenges associated with the delivery of cells by these methods. These delivery methods are only suitable for a small proportion of the possible cell therapies. Many other treatments under development for illnesses
Cell numbers, viability and frequencies pre and post CD56 enrichment.
Validation Pre Proccess
CD56+ Fraction
1 2 3 4 5 6 Median 1 2 3 4 5 6 Median
Viability (%)
TNC (x106)
% CD3+
CD3+ (x106)
% CD56+
CD56+ (x106)
%CD56+ CD3+
CD56+CD3+ (x106)
82.70 91.50 90.50 88.10 98.75 51.00 89.30 96.70 59.40 97.40 96.30 98.75 99.10 97.05
1099.18 947.58 839.16 1342.70 1213.20 11234.70 1156.19 57.72 47.56 93.60 126.80 104.00 348.50 98.80
65.00 47.10 64.50 44.50 68.20 59.40 61.95 3.76 5.51 1.30 3.61 5.72 1.38 3.69
537.28 398.57 367.04 381.11 567.04 2880.00 467.93 1.99 1.55 1.06 4.28 5.42 4.70 3.14
13.10 10.30 16.70 17.00 10.00 12.10 12.60 76.20 72.80 93.90 82.60 69.30 84.80 79.40
108.28 87.16 95.12 145.59 83.14 568.50 101.70 40.23 20.48 76.53 97.84 65.72 289.10 71.13
2.09 1.78 0.79 2.26 3.67 1.20 1.94 7.66 17.30 3.05 10.10 23.00 11.40 10.75
17.28 15.06 4.50 19.36 30.51 59.20 18.32 4.04 4.87 2.49 11.96 21.81 38.90 8.42
Abbreviations: TNC - Total nucleated cells. Phenotypic analysis consisted of gating on forward scatter vs. side scatter to eliminate cell debris and a second gate on viable cells using TO-PRO-3.
Poster Abstracts
control strategy developed. MSC identity and quality characteristics have been assessed following our novel harvesting and cryopreservation process with MSCs demonstrating full ISCT criteria as well as maintaining monolayer outgrowth and colony-forming efficiency. Increased inter-donor consistency of MSC product has been demonstrated in long term serum-free culture in terms of growth, metabolomics, colony-forming efficiency, osteogenic potential and expression of key genes involved in immunomodulatory and angiogenic function. These findings have implications in the development of both autologous and allogeneic cell therapy bioprocesses, with consistency gains having great potential to provide significant cost savings in process development and validation. This convergence in product yield and quality has been driven by the systematic development of a controlled, scalable and serum-free MSC expansion, harvest and cryopreservation process.
Figure 1. Driving consistency into the MSC expansion process. (A) increased consistency in MSC growth for serum-free media (SFM) compared to bovine-serum (FBS) and human-serum (HPL) between multiple donors & (B) increased consistency in MSC quality attributes in SFM between donors. 283 WILL NOT BE PRESENTED 284 IMPLEMENTATION OF ACADEMIC ADVANCED THERAPY MEDICINAL PRODUCTS IN CLINICAL PRACTICE IN THE NETHERLANDS Sd Wilde1, LA Veltrop-Duits1, M Hoozemans-Strik2, J Veenman3, H-J Guchelaar1, M Zandvliet1, P Meij1 1 Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, Netherlands, 2Dutch Cancer Society, Amsterdam, Netherlands, 3Netherlands Research Organisation of Health Research and Development (ZonMw), The Hague, Netherlands Advanced Therapy Medicinal Products (ATMPs), including gene therapy products (GTMPs), somatic cell therapy products (sCTMP) and tissue engineered products (TEP), can only be applied under the European regulatory framework, (1) within a clinical study protocol, (2) within the hospital exemption (HE) and (3) with an EMA (European Medicines Agency) Marketing Authorization. Until now only four ATMPs (ChondrocelectÒ, GlyberaÒ, MACIÒ and ProvengeÒ) succeeded to achieve EMA approval, despite the fact that a lot of ATMPs were tested in clinical trials. Most ATMPs developed in academia, which appear to be safe and effective, remain in developmental phase. This project aims to identify how academic ATMPs can efficiently become available for regular clinical care. The association between variables and success or failure in the route towards implementation in regular care will be analyzed. From different academia in The Netherlands 49 ATMPs** were selected. Variables that could have impact on either the development or the implementation of the products are analyzed using interviews with research groups and stakeholders. Financing is the main hurdle in ATMP development. The intention to register an ATMP is clear for the GTMP projects in contrast to most CTMP projects. Intellectual property (IP) has been secured in 52% and HE was used in 17% of the products with registration intention. In 64% of the ‘uncertain about registration group’ proof of efficacy is considered essential before thinking about further implementation in regular clinical care. The impossibility to further modify a product after registration is for research groups a motive to not registrate their product. Based on the results, optimal routes for academic development and implementation of ATMPs will be designed and communicated to the academic field.
** This project and the selected ATMPs are supported by Dutch Cancer Society (DCS) and Netherlands Research Organisation of Health Research and Development (ZonMw). 285 OPTIMIZED MANUFACTURING PROCESS FOR THE GENERATION OF CLINICAL GRADE CAR T CELLS P Bajgain1, R Mucharla1, N Watanabe1, J Wilson2, U Anurathapan1, N Lapteva1, H Heslop1, C Rooney1, M Brenner1, AM Leen1, J Vera1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, Houston Methodist Hospital, Houston, Texas, United States, 2Wilson Wolf Manufacturing, St Paul, Minnesota, United States Although adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has shown promising clinical responses, prolonged and complicated cell production process has limited its broader application. To simplify CAR T cell manufacturing, we assessed whether cell expansion could be improved by: (i) supplementing cultures with artificial antigen presenting cells (a-APC), and (ii) growing cells in a gas permeable culture device (G-Rex). As a proof of concept, CAR T cells targeting the prostate cancer antigen, PSCA. By retroviral transduction of K562 cells to express PSCA, we generated an a-APC cell line, which resulted in a 3.4 fold greater CAR T cell expansion relative to IL2 alone. Moreover, CAR-T cells were enriched in the a-APC cultures, increasing from 43.47.6% to 85.59.2% (n¼3) in 10 days, unlike IL2-expanded cells where they remained unchanged (43.37.6% to 43.44.1% on day 10). Although this a-APC-based approach induced selective CAR-T cell expansion, using traditional 24-well tissue culture-treated plates made the manufacturing process labor and time intensive. Thus, to simplify cell production, we assessed whether CAR-PSCA T cells with a-APC stimulation(1:2) could be expanded in a static GMP-compliant G-Rex 100M with a surface area of 100cm2. From an initial 25E+06 CAR T cells, we obtained 2963.8195.2E+06 cells (n¼3) in 10 days, a 118.6 fold increase using only 1 liter of media. As expected, the expanded population were enriched for transgenic CAR-T cells (43.47.6% - day 0 and 83.67.0% - day 10; n¼3). Importantly, T cells manufactured using this method were able to better retain anti-tumor activity and expression of memory and activation markers such as CD62L and CD25 when compared to cells maintained in conventional culture. In order to further simplify the manufacturing process, we have now developed a semi-automatic cell collection device that can be paired G-Rex 100M, which allows cell collection in a small volume (100ml) in under five minutes. 286 OPTIMIZATION OF XENO-FREE EXPANSION CONDITIONS FOR HUMAN UMBILICAL CORD-DERIVED PERIVASCULAR CELLS USING FUNCTIONAL IMMUNOPHENOTYPING AND IN VITRO POTENCY ASSAYS M Librach1, S Pereira1, L Maghen1, F Iqbal1,2, T Barretto1, K Park1, P Szaraz1,2, A Gauthier-Fisher1, C Librach1,3,2,4 1 CReATe Fertility Centre, Toronto, Ontario, Canada, 2Department of Physiology, University of Toronto, Toronto, Ontario, Canada, 3Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada, 4Department of Gynecology, Women’s College Hospital, Toronto, Ontario, Canada Xeno-free expansion of MSCs is important for their clinical translation to cell therapy. Recent reports demonstrated the maintenance or enhancement of proliferative potential, immunological and MSC properties in xeno-free culture conditions, yet very few studies assessed the expression of molecules and phenotypes that could impact the acute behavior of MSCs post-implantation. We isolated and expanded first trimester human umbilical cord-derived perivascular cells (FTM-PVCs) in 4 commercially availabe xeno-free human MSC culture media(A-D), as well as serum containing conditions(aMEM + 10% FBS). Media A did not support the expansion of FTM-PVC cultures. Only media B and aMEM + 10% FBS conditions supported the establishment of FTM-PVC cultures. We compared the expansive capacity and population doubling time (PDT) through 9 passages. With the exception of media C, which showed drastically reduced PDT at early passages, the other 3 zeno-free conditions were comparable to FBS-containing conditions. We assessed the immunophenotype of expanded FTM-PVCs at passages 3 and 6 using flow cytometry for surface markers that define MSCs. The majority of cells were CD90+ and CD44+ in all conditions. CD105 expression decreased in media C and D conditions. However, we observed that the expression of markers
21st ISCT Annual Meeting
associated with immunoprivilege(HLA-G), with acute responsiveness(CXCR4, CD49f, PDGFRbeta) or other cellular attributes(SSEA4, CD146, Oct4A, MMPs) varied dramatically between culture conditions, with media B giving expression levels closest to serum conditions for the majority of these markers. In vitro functional potency assays evaluated and compared the immunosuppressive, chemotaxis, extravasation and angiogenic properties of FTM-PVCs expanded in each condition. Our results suggest that while MSC properties are maintained in most xeno-free conditions, characteristics that could significantly impact acute behavior in vivo vary greatly between the culture conditions evaluated. 287 VALIDATION OF THE CliniMACS PRODIGY FOR MANUFACTURE OF TUMOR-PRIMED NATURAL KILLER CELLS (TpNK) H Bartley1, L Clarke2, M Lowdell1, ER Samuel1 1 Haematology, University College London, London, United Kingdom, 2Miltenyi Biotec, Bisley, United Kingdom The production of clinical-grade TpNK cells under good manufacturing practice (GMP) includes immunomagnetic isolation of NK (CD56+/CD3-) and NKT (CD56+/CD3+) cells, priming with tumor-lysate (TL) and removal of contaminating TL, QC testing and final release. Current methods result in CD56+ yields of w30% with purity of >95% but with significant TpNK loss following TL removal by density gradient separation in an open-system. This study aimed to validate the translation of TpNK manufacture onto the CliniMACS Prodigy to evaluate NK cell yield and purity and efficiency of TL removal within a closed-system. CD56+ NK cell isolation on the CliniMACS Prodigy was assessed in six donor leukocyte apheresates and TL removal experiments performed using the CTV-1 cell line as mock TpNK cells, co-cultured with TL in three validation runs. CD56+ enrichment and TL removal were performed using an enrichment program 2.0 and a lysate-ficol program respectively. CD56+ isolation resulted in a median purity of 92.3% (83.9-97.0%), yield of 66.5% (35.3-79.3%) and a median CD3+ T cell log depletion of 2.4 (2.0-2.5). Median viability was 96.7% (68.3-98.1%) and total processing time was 140 minutes. Assessment of TL removal by ficol-density gradient resulted in a median CTV-1 yield of 59.0% (48.0-68.0%), TL depletion of >90% and a median viability of 99.0% (95-99%), meeting acceptance criteria for GMP manufacture and TpNK cell final release. TpNK cell manufacture on the CliniMACS Prodigy is feasible, demonstrating improved CD56+ yields following isolation and improved recovery after TL removal. This will substantially reduce COGs and increase the TpNK cell dose from a single donor apheresis. Translation to a fully closed-process on the CliniMACS Prodigy will allow transfer of manufacturing into a Grade D GMP suite from a current Grade B laboratory achieving significant savings in GMP operating costs.
S83
288 PRENATAL AND POSTNATAL TRANSPLANTATION OF HUMAN AMNIOTIC FLUID STEM CELLS IN SPINAL MUSCULAR ATROPHY MICE S Shaw1,2, L-K Tsai3, P-J Cheng1 1 Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taoyuan, Taiwan, 2Institute for Women’s Health, University College London, London, United Kingdom, 3National Taiwan University, Taipei, Taiwan Human amniotic fluid is a promising resource of pluripotent stem cells and it makes fetal therapy using autologous transplantation possible. Human amniotic fluid stem (AFS) cells are a unique subgroup of human AF-derived stem cells that are isolated by C-Kit immunoselection, and which possess amazing capability of self-renewal and potential to differentiate. Several human stem cells have engrafted successfully in fetal mice via in-utero transplantations (IUT) that include the stem cells from fetal blood and bone marrow, but amniotic fluid yet. We designed IUT into spinal muscular atrophy (SMA) mice using human AFS cells. Adult SMA mice had been proved that could be treated with human AFS by other research group. We transplanted 100,000 human AFS cells per pup into the fetal mice (type I and type III SMA) intraperitoneally at E13 and 3 months postnatally. There were 89 fetuses from 9 dams received prenatal cell therapy with 62% survival rate. Three experimental groups were designed as human AFS cells, human fibroblasts or normal saline injection into fetuses for comparison. The preliminary results showed the engraft evidence in the PCR, flowcytomery and immunohistochemistry among multiple fetal organs especially in the skeletal muscles. However, we did not see the prolong survival for type I SMA (severe form) yet after in utero human AFS cells transplantation. The functional test of long-term survival in type III SMA showed significant improvement compared to untreated group with longer survival. This is the first study using human amniotic fluid stem cells as cell therapy source for prenatal transplantation into mice SMA model. For the clinical purpose, these cells either with mesenchymal or hematopoietic potential could be obtained prenatally for autologous cell fetal therapy, or storage the cells for postnatally tissue engendering in the future. 289 OVERCOMING THE TRANSLATIONAL CHALLENGES OF THE EFFECTIVE ADMINISTRATION AND DELIVERY OF CELLS A Lyness, N Medcalf, D Williams Wolfson School of Mechanical & Manufacturing Engineering, Loughborough University, Loughborough, Leicestershire, United Kingdom Of the few cell-based therapies widely available today the most mature are the treatment of blood-borne cancers and wound healing. The routes of administration for these applications are well understood and a great deal of research has resolved the many biological, pharmacological and engineering challenges associated with the delivery of cells by these methods. These delivery methods are only suitable for a small proportion of the possible cell therapies. Many other treatments under development for illnesses
Cell numbers, viability and frequencies pre and post CD56 enrichment.
Validation Pre Proccess
CD56+ Fraction
1 2 3 4 5 6 Median 1 2 3 4 5 6 Median
Viability (%)
TNC (x106)
% CD3+
CD3+ (x106)
% CD56+
CD56+ (x106)
%CD56+ CD3+
CD56+CD3+ (x106)
82.70 91.50 90.50 88.10 98.75 51.00 89.30 96.70 59.40 97.40 96.30 98.75 99.10 97.05
1099.18 947.58 839.16 1342.70 1213.20 11234.70 1156.19 57.72 47.56 93.60 126.80 104.00 348.50 98.80
65.00 47.10 64.50 44.50 68.20 59.40 61.95 3.76 5.51 1.30 3.61 5.72 1.38 3.69
537.28 398.57 367.04 381.11 567.04 2880.00 467.93 1.99 1.55 1.06 4.28 5.42 4.70 3.14
13.10 10.30 16.70 17.00 10.00 12.10 12.60 76.20 72.80 93.90 82.60 69.30 84.80 79.40
108.28 87.16 95.12 145.59 83.14 568.50 101.70 40.23 20.48 76.53 97.84 65.72 289.10 71.13
2.09 1.78 0.79 2.26 3.67 1.20 1.94 7.66 17.30 3.05 10.10 23.00 11.40 10.75
17.28 15.06 4.50 19.36 30.51 59.20 18.32 4.04 4.87 2.49 11.96 21.81 38.90 8.42
Abbreviations: TNC - Total nucleated cells. Phenotypic analysis consisted of gating on forward scatter vs. side scatter to eliminate cell debris and a second gate on viable cells using TO-PRO-3.