Clinical Applications of Mesenchymal Stem Cell Therapy

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1 Clinical Applications of Mesenchymal Stem Cell Therapy Dr R R Sharma MD Professor Department of Transfusion Medicine Postgraduate Institute of Medical Education and Research, Chandigarh, India

2 What are Stem Cells? Stem Cells are extraordinary because: They can divide and make identical copies of themselves over and over again (Self-Renewal) Remain Unspecialized with no specific function or become.... Specialized (Differentiated) with the potential to produce over 200 different types of cells in the body.

3 Stem Cells are like magnets Society Research Institutions Media Investors Public/Private Stem Cell Technology biotechs Stem Cell Academic leaders Regenerative Medicine Pharmaceutical Industry Equipment Providers Cryobiologists

4 Basis of Stemness Asymmetric Division Daughter stem cell Daughter transit-amplifying or intermediate cell Repopulates stem cell pool Proliferative, Migratory and Differentiating capabilities Intermediate cell progenitors Mature tissue

5 Maintenance of Stemness The length of the telomere determines the ability of the stem cell to keep from differentiating and aging. Telomeres contain highly conserved G-rich repeats Thus, stem cells contain a lot of telomerase.

6 Sources of Stem Cells Totipotent Pluripotent Multipotent : Zygote, Morula : Embryonal stem cells (inner cell mass) : Amniotic epithelical cells : Fetal stem cells : Umbilical cord stem cells : Adult stem cells : Fetal stem cells : Umbilical cord stem cells : Adult stem cells - bone marrow, peripheral blood, brain, eyes, heart, lungs, kidneys, GIT, pancreas, liver, fat, breast, ovaries, prostate, testes

7 Adult Stem Cells Hematopoietic Stem Cells Bone marrow (1% to 3%) Mobilized peripheral blood (0.01% to 1%) Umbilical cord blood Mesenchymal Stem/stromal Cells Bone marrow (0.0001% to 0.01%) Umbilical cord blood (0.0001% to %) Placenta (better source) Tissue specific

8 Mesenchymal Stem /stromal Cells 1 st described in 1968 (Friedenstein) Adherent, clonogenic, fibroblastic marrow cells Multiple sources ISCT definition (2006) Plastic adherence CD73, CD90, CD105 (+); lineage markers (-) In vitro differentiation to bone, fat, cartilage Lack of HLA-Class II Antigens(Immunopriviliged )

9 Mesengenesis Adult Mesenchymal Stem Cell (MSC) Osteoblast Chondrocyte Myoblast Fusion Stromal Fibroblast Tenoblast Preadipocyte BONE CARTILAGE MUSCLE STROMA TENDON ADIPOSE

10 The global landscape of stem cell clinical trials ClinicalTrials.gov & WHO s International Clinical Trials Registry Matthew D Li, Harold Atkins Regen. Med. (2014) 9(1), 27 39

11 Matthew D Li, Harold Atkins Regen. Med. (2014) 9(1), 27 39

12 Database of clinical trials (n = 4749) Novel (n = 1058) Non-novel (n = 3961) Goal of stem cell therapy Regeneration (n=916) Cell therapy (nonregenerative) (n=126) Gene therapy (n=96) Stem cell collection/mobilization (n=30) Bioscaffold (n=15) Immunotherapy (n=13) Stem cell type Hematopoietic (whole marrow, CD34+, D133+ or mononuclear fractions) (n=432) Mesenchymal (n=432) Endothelial progenitor cells (n=69) Other (n=69) Neural (n=22) Unspecified (20) Limbal (16) Embryonic (6) Cardiac (6) Principle disease/condition targeted Cardiovascular disease (n=278) Neurological disease (n=169) Cancer (n=97) Liver disease (n=67) Bone condition (n=65)) Other (n=56) Immunodeficiency and other nonmalignant hematologic conditions (n=49) Gastrointestinal disease (n=46) Cartilage disease (n=45) Systemic rheumatological disease (n=45) Diabetes (n=43) Eye disease(n=39) Skin condition (n=19) Organ transplant-associated (n=18) Lung disease (n=15) Kidney condition(n=8)

13 stem cell-related therapy and product development are likely to be an $8.5 billion global market by 2016 Matthew D Li, Harold Atkins Regen. Med. (2014) 9(1), 27 39

14 Therapeutic Potential of Mesenchymal Stem/stromal Cells 1. Tissue repair and regeneration as they differentiate into many tissues 2. Immunomodulation 3. Enhancement of HSC engraftment 4.Remarkable expansion after ex-vivo culture, even with platelet lysate and maintain genetic stability

15 Biological Properties of MSCs

16 Paracrine Mechanisms of Mesenchymal Stem Cell-Based Therapy Cell Transplantation, Vol. 23, pp , 2014

17 Instant blood mediated inflammatory reaction (IBMIR)

18 MSC engraftment, antagonizing mechanisms, and differential priming of therapeutic effects TIME IN HOURS

19 Clinical Applications ( ) June 2015 (453 Clinical Trials) 15% 20 % 13% 19% 15% 18%

20 Source of MSC S ( ) June 2015 (453 Clinical Trials)

21 MSCs Isolation Bone Marrow Aspiration Isolation Culturing Administration Final Product

22 MSC Isolation from Bone Marrow ml BM Aspiration PSIS under LA MNC separation by Ficoll density gradient Cell seeding (5x10 4 MNC/cm 2 to 1.7 x 10 5 cells/cm 2 ) at different conc. in MEM (with & without Fetal bovine serum) 50% of Medium & Non-adherent cells will be replaced with fresh medium Procedure 3 days until 70-80% confluency in the adherent cells Cell replating (1-5) x 10 3 MSC s/cm 2 (in different conc.) to (1-5)x10 5 MSC s/cm 2

23 QC assessment Cell counts Automated cell counters Viability Trypan blue dye exclusion/acridine orange/propidium iodide ( 70%) Immunophenotyping - CD105>75%, CD73>85%, CD-90>85% CD-34 & 45 (<5%) Trilineage differentiation assay - Osteo, chondro and adipogenic lineages Aerobic/Anaerobic bacterial cultures, fungal cultures Mycoplasma testing Endotoxin Assay <5 EU/kg/dose Dose of MSC s-- 1X10 6 cells /Kg

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25 hmsc Day 8 (10X) Day 16 (10X) Day 24 (10X) Adipocyte differentiation Control (10X) 10X After differentiation 20X

26 Osteocyte differentiation Control (10X) 10X After differentiation 20X Cardiomyocytes differentiation Before differentiation (10X) Control undifferentiated (40X) Actinin (40X) Troponin (40x) Differentiated MSC

27 Characterization of MSC Negative CD34-PE CD45-FITC Positive CD105- FITC CD90-PE CD73-PE Fig: A- Unstained(US) MSC Fig: B- MSC showing positive for Stemness marker Fig: C- MSC negative for Hematopoietic lineage marker A B US 97% 97% US 90%

28 MSCs Clinical Studies Done at Stem Cell Research Facility PGIMER,Chandigarh Autologous Bone Marrow Mononuclear Cells In Idiopathic Membranous Nephropathy Results: Autologous MNC cell infused to patients with biopsy proven IMN showed a transitory reduction in proteinuria and improvement in serum albumin in treatment refractory IMN. Effect Of Mesenchymal Stem Cells On The T Cell Repertoire Of Kidney Transplant Patients Results: T cell proliferation is markedly reduced with MSC therapy at various time points post transplantation. T-Cells that generally progress through six divisions using CFSE dilution assay were arrested following two divisions at 90 days post transplantation. Trans-differentiation of cultured Mesenchymal Stem Cell into islets cells Results: Evaluated the therapeutic effects of MSC (2x10 6 /kg) infused intravenously to streptozotocin induced diabetic wistar rats, two times at day 7 and 21 of diabetic induction, reported decreased sugar levels.

29 Indian J Med Res 142, July 2015, pp DOI: /

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31 Adult mesenchymal stem cells and chronic burn wound epithelialization Results: Autologous bone marrow derived mesenchymal stem cell transplanted to, two areas similar in size and depth of burns, followed by split skin grafting. The test wounds healed completely at an earlier date compared to the control wounds in six patients. Mesenchymal stem cell and Osteoarthritis Results: Intra-articular injection of autologous MSCs can be considered a potential treatment of early osteoarthritis knee which relieves pain, stiffness, improves physical functions and improves the articular cartilage integrity to relief treatment of OA. Autologous mesenchymal stem cells and meniscal tear of knee joint Results: Autologous MSC infused to patients showed a transitory reduction in pain and improvement in meniscal tear. Human dental pulp stem cells (DPSCs), umbilical cord, blood Techniques used: isolation of dental pulp, follicle and apical papilla, expansion, characterization and differentiation to adipocyte and osteocytes.

32 Challenges in Clinical Medicine 1. What SCs to use? 2. At what stage of the disease will therapy be most effective? 3. What dose, which site? 4. Single or multiple doses? 5. Regulatory Framework

33 Donor Related Issues Requirements similar to other tissue based products(allogeneic Bone Marrow donors) Stringent Donor screening for infectious & genetic disease testing (one donor-multiple recipients) Age of Donor(10-fold birth teenage &10-fold teenage -elderly

34 Stem cell based therapeutics large scale manufacturing Regulatory requirements biological license application Establishment of safety and efficacy Investigational new drug application Compliance with GMP/GTP

35 Regulatory Issues Stem cell use in patients, other than that for hematopoietic stem cell reconstitution for approved indications, is investigational at present. Accordingly, any stem cell use in patients must only be done within the purview of an approved and monitored clinical trial with the intent to advance science and medicine, and not offering it as therapy. In accordance with this stringent definition, every use of stem cells in patients outside an approved clinical trial shall be considered as malpractice.

36 Regulatory Issues Institutional committee on Stem cell research (ICSCR)&Institutional Ethics Committee (IEC) National Apex Committee for Stem Cell Research and Therapy (NAC-SCRT) All Pre-clinical/Clinical trials SCR must be registered with ICMR Clinical Trial Registry

37 Future Directions in MSC Research Need to identify an ideal source Optimization Culture, characterization and cryopreservation Techniques Understanding of the biology of Paracrine effects Defining appropriate cell dose and frequency Appropriate Donor Selection Working as per the regulatory frame work

38 Churning of the Ocean. For the Nectar of Immortality

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40 Matthew D Li, Harold Atkins Regen. Med. (2014) 9(1), 27 39