Mesenchymal Stromal/Stem Cells

Mesenchymal Stromal/Stem Cells Peiman Hematti, M.D. Department of Medicine Financial Disclosure: Consulting Fees from Celgene Cellular Therapeutics in 2011 & 2012 1

Caplan used the term Mesenchymal Stem Cells in 1980 s due to their multi-lineage differentiation potential (bone, fat, cartilage, marrow stroma, ) Terminology has evolved Terminology has evolved Colony forming units-fibroblasts (CFU-E) Fibroblast colony forming cells (FCFC) Marrow stromal cells Mesodermal progenitor cells Mesenchymal stem cells y Multipotent marrow stromal cells Mesenchymal stromal cells Mesnechymal stromal/stem cells 2

The stem cell niche in bone marrow JCI Tong Yin, et al. 116:1195 May 2006 The concept of HSC niche is rapidly evolving 4

Derivation of MSCs from Bone Marrow Clinical Applications Basic Research The first report on clinical use of ex vivo culture expanded MSCs 5

The first report on clinical use of ex vivo expanded autologous MSCs with HSCs The first report on clinical use of ex vivo expanded allogeneic MSCs with HSCs 6

Anecdotal reports on successful use of MSCs for reversion of cytopenias after HSCT The first report on use of allogeneic MSCs for nonhematological disorders 7

MSC Plasticity!!! http://stemcells.nih.gov/info 8

Prochymal has been approved only in Canada and New Zealand for Pediatric GVHD Promising results but most studies are small and nonrandomized 14

MSCs in tissue engineering 15

Bone Marrow Adipose Tissue Placenta Umbilical cord Muscle Heart Lung Pancreas Liver Kidney Bladder Tooth Synovia Ligament GI Tract Stem Cells, Volume 26, Issue 9; Pages 2287-2299 Proposed model of the MSC role in the tissue repair process. 16

Adipose tissue-derived MSCs Gimble, J. M. et al. Circ Res 2007;100:1249-1260 17

Why MSCs are so popular? Allogeneic MSCs can be used as an off the shelf cellular therapeutics Systemic administration of MSCs have shown to be safe No major adverse reaction has been reported No ectopic tissue formation has been reported No tumor formation has been reported in clinical trials Local injection of MSCs, even into spinal cord or brain, is safe Permanent engraftment may not be needed for efficacy MSCs have a multitude of effects Immune modulatory Anti-inflammatory Angiogenic properties Growth factor release 19

UW-Madison PACT Project-MSC To generate MSC banks for translational research projects, pre-clinical animal studies, and human clinical trials MSCs for Bronchiolitis obliterans after lung transplantation (Zubair) MSCs for Myocardial Infarction (Raval) MSC Culture Media Evaluation FBS standard vs. irradiated, lot variability StemPro (Invitrogen) MesenCult (Stem Cell Technologies) Quality Control assay development Potency assay development need several assays to determine impact of culture method on MSCs Development of cgmp documentation for standard test methods Factors to consider in MSC culture/utilization Staring tissue material: BM vs. fat Autologous vs. allogeneic Processing: Ficoll, RBC lysis, direct plating, Plating density Passaging procedure (timing, i plating density, ) Culture media FBS vs. no FBS containing media Irradiation of FBS, lot of FBS and concentration of FBS Use of alternatives such as platelet lysate Coating matrix Freezing methodology Passage # to be used in the clinic Cell dose and frequency of administration Potency assay (indication specific?) Immune modulatory/anti-inflammatory properties Angiogenic potential Paracrine effects 20

There is no standard culture methodology for MSCs PACT#1 Culture method BM volume: ±25 ml (fresh or frozen) MNC prep: Ficoll Medium: α-mem, 16.5% FBS, 1% GlutaMax Medium changes, frequency: days 1 and 2 to wash out non-adherent cells; then every 2-4 days Passes, length of culture: after 70-80% confluence at day 7-10, cells are inoculated into a cell factory at 40-50 cells/cm2. On day 18-21 at 70-80% confluence, cells are harvested for final prep Characterization: Flow cytometry: Positive for: CD73, CD90, CD105; Negative for : CD14, CD19, CD34, CD45, HLA-DR Functional/Potency Assays: Trilineage: Yes Suppression (NLR, CFSE, cytokines): Yes (limited) Other cytokines: N/A CFU: Initially attempted, but abandoned PACT#2 Culture method BM volume: ±10 ml MNC prep: None, direct plating of whole BM Medium: α-mem, 10%FBS, 5ng/ml bfgf Medium changes, frequency: initially every 2-3 day to wash out non-adherent cells none once subculture Passes, length of culture: initial culture lasts about 10-14 days, then we passage every 5-7 days Characterization: Flow cytometry: Positive for: CD90, CD73, CD29, CD105, CD166, HLA-ABC, Negative for : CD34, CD45, CD31 Functional/Potency Assays: Trilineage: only to validate the protocol originally Suppression (NLR, CFSE, cytokines): N/A Other cytokines: N/A CFU: N/A Other: depends on the project; the potency assay is specific/adapted to the function of the product PACT#3 Culture method BM volume: 25-100 ml; fresh MNC prep: Ficoll, RBC lysis Medium : α-mem, 10% FBS, 1% Glutamax Medium changes, frequency: Every 2-4 4days; to 8090% 80-90% confluence Passes, length of culture: cryopreserved at passage 2 (P2); Final clinical MSC product typically is designated passage 5 (P5) Characterization: Flow cytometry: Positive for: CD73, CD90, CD105 Negative for: CD34, CD45, CD14, CD19, HLA-DR Functional/Potency Assays: Trilineage: only to validate the protocol initially Suppression: CFSE-based MLR in the process of converting to Elisabased sil-2r Other cytokines: N/A CFU: N/A Other: Clinical indication specific Generation of MSCs: UW-PACT Evaluation of different culture media FBS StemPro MesenCult % Proliferation in CD4+ gate PBMC-2, Passage 2 100 80 % CFSE-lo 60 40 20 StemPro FBS-Irr 0 1-0 P-M 1-1 P-M 1-0.5 P-M 1-0.25 P-M 1-0.1 P-M 1-0.05 P-M PBL:MSC Ratio 21

Goals: Potency assay development To develop potency assays for comparison of MSCs generated via different culture methodologies, from different sources, different passages, Identify a potency assay that is predictive of efficacy outcome (FDA expectation to be developed by phase-iii trials) Assay requirements: Easy, reproducible and practical to be performed by different (PACT) centers Comprehensive, if possible Unresolved questions: Need for animal models? Can any in vitro assay mimic MSC potency in vivo? Is it possible to develop an assay that is predictive of clinical outcome? Different potency assays for different indications? Practical and reproducible potency assay for phase III trials? Clinical Trials of MSCs Enhancement of HSC engraftment Autologous, Allogeneic HSCT Ex vivo expansion of cord blood CD34+ cells Treatment of congenital disorders Osteogenesis Imperfecta Metachromatic leukodystrophy, Hurler syndrome Immune modulation Graft versus host disease Prevention/treatment of rejection after solid organ transplant Autoimmune disorders (lupus, systemic sclerosis) Tissue regeneration Heart repair Acute myocardial infarction, chronic ischemia, heart failure COPD Crohn s disease Neurological disorders Amyotrophic lateral sclerosis, stroke, spinal cord injury, multiple sclerosis Diabetes Cirrhosis Acute kidney Injury Lower extremity ischemia Tissue repair after HSCT (perforated colon, pneumothorax, hemorrhagic cystitis) Tissue reconstruction Wound repair, fistula repair, soft tissue reconstruction Tracheal repair 22

How about Macrophages?? Antonio Uccelli, Lorenzo Moretta & Vito Pistoia Human MSC-Educated Macrophages: IL-10, IL-12, IL-6, TNF-α A Novel type of Alternatively Activated Macrophages Macrophages derived from PB monocytes ( CD14+ cells cultured x 7 days) MQ+ MSC MQ BM-MSCs co-cultured with MQs x 3 days Immunophenotype of macrophages determined MQ+ MSC MQ MQ+ MSC MQ MQ+ MSC MQ 23

MSCs increase expression of CD206 on MQs A potential universal potency assay? Thank You Funding: NIH, NHLBI NIH, NHLBI, PACT Award Department of Defense National Blood Foundation Stem Cell Research Foundation UW-Carbone Cancer Center 24