Cell-based therapies in tissue engineering and regenerative medicine (TE/RM)
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1 Cell-based therapies in tissue engineering and regenerative medicine (TE/RM) Pamela Gehron Robey, Ph.D. Craniofacial and Skeletal Diseases Branch National Institutes of Dental and Craniofacial Research Co-coordinator, NIH BMSC Transplantation Center Acting Director, NIH Stem Cell Unit National Institutes of Health Department of Health and Human Services 8th Annual National Summit on Health Disparities April 11 th, 2011 I am a US Government employee. I have no conflicts of interests.
2 Points to be covered: stem cell definitions and development stem cells and their use in regenerative medicine adult bone marrow stromal stem cells as an example of the process (many others under development) current activities at IRP NIH in cell-based therapies
3 Properties of stem cells in the body: offspring of a single cell are able to reconstitute an entire tissue able to self-renew (maintain a stem cell in the population after cell division)
4 Development of stem cells blastocyst gastrulation - three germ layers fertilized egg inner cell mass ectoderm mesoderm endoderm toti-potent pluri-potent (cells of the embryo, not cells of placental membranes) ES cells induced pluripotent cells (ips cells) factors multi-potent fetal stem cells, lineage committed birth adult stem cells differentiated cells
5 What we MUST know about stem cells to use them in TE/RM embryonic stem cells unlimited replication pluripotency ipscs fetal stem cells multipotency lineage commitment adult stem cells controlled and predictable differentiation
6 Adult bone marrow: home of a dual system of stem cells fat bone blood blood stem cells bone marrow stromal ( mesenchymal ) stem cells
7 A single bone marrow stromal stem cell regenerates all types of skeletal cells A single colonyforming unit-fibroblast (CFU-F) micromass culture cartilage in vivo transplant ossicle forms a colony Bianco et al., Stem Cells, 2001 bone cells, marrow fat cells, cells that support blood formation
8 BMSCs in TE/RM direct orthotopic transplantation optimize composition, size and shape of vehicle percutaneous delivery of cells with an injectable carrier injury or lesion ex vivo expansion (must maintain stem cells) molecular engineering cell surface character escape from circulation systemic injection? role of vasculature integrity and injury sufficient numbers for biological function
9 Craniofacial reconstruction with BMSCs critical size calvarial defect mandibular onlay Mankani et al., Am J Pathol, 2006; Mankani et al., Stem Cells, 2006
10 Systemic injection: treatment of diseases BMSCs do not form non-skeletal cells or persist for long periods of time neuronal diseases lung fibrosis cardiovascular disease there is a beneficial effect on injuries and in disease states BMSCs may nurse local cells to begin the repair process by secretion of growth factors liver disease kidney disease skin ulcers pancreas (diabetes) inflammatory bowel disease immunomodulation immunosuppression
11 NIH Bone Marrow Stromal Cell Transplantation Center Mission: build the infrastructure necessary to produce high quality clinical grade BMSCs that maintain their biological activities (applicable to many cell types) assist clinical investigators in the preparation of clinical protocols for IRB approval, and regulatory documents (Drug Master File and INDs) for submission to the FDA (cross the Valley of Death )
12 Development of cell-based therapies Code of Federal Regulation for Food and Drugs (21 CFR 600 Biologics) Donor evaluation, consent, tissue acquisition regulations vary depending on autologous, allogeneic (related), or third party, and clinical usage (bone marrow aspirates) Cell processing and product characterization sterility, identity, purity (composition), potency and stability (testing, cell surface markers, mixed lymphocyte reaction, storage) Evaluation of cell product appropriate animal models, biodistribution, stability, safety (toxicity, tumorigenicity), efficacy (in vivo transplants)
13 Cell factories Semi-closed system: have modified commercially available system
14 Bioreactor - prototype 16x10 6 cells plated in the bioreactor; harvest on day 7=206x10 6 cells
15 Current status Donor Evaluation and Marrow Acquisition Protocol developing a BMSC repository Drug Master File approved by FDA refractory acute GVHD (Barrett, NHLBI) (IRB approval pending, IND about to be approved) refractory inflammatory bowel disease (Yao, NIAID) ischemic coronary disease (Horvath, NHLBI) bone regeneration (Robey, NIDCR) radiation induced injury (Citrin, NCI) and others
16 Mission: characterize approved hes cell lines establish standards for the culture process, as well as quality control and monitoring of these hes cell lines create a database for comparison of hes cell lines, adult stem cells and ips cells now working on ipscs provides support to intramural investigators
17 hes cells: bone formation in vivo HSF-6 scaffold human osteocyte bone human osteoblast determine conditions for bone differentiation introduce known mutations in order to develop a more continuous source of human mutated cells for study need to to develop more consistent bone differentiation conditions now applying this condition to induced pluripotent cells (IPCs) Kuznetsov et al., Stem Cells & Dev, 2011
18 ipscs - osteogenic differentiation ipscs from BMSCs - monolayer mesodermal cells in vivo transplantation sequential morphogen, GF treatment osteogenic conditions stay tuned calcification in vitro (alizarin red staining)
19 Summary Embryonic stem cells Induced pluripotent stem cells Fetal stem cells Adult stem cells Derivation inner cell mass of the blastocyst viral transduction or by small molecules of adult cells tissue-specific protocols from aborted fetuses tissue-specific protocols from different tissues Advantages unlimited division, pluripotent, no molecular alteration unlimited division (?), pluripotent, no destruction of a blastocyst, autologous extensive division, committed to lineages moderate division, commitment to specific cell types, autologous Disadvantages destruction of embryo, incomplete differentiation, possibility of teratoma, allogeneic viral integration, equivalence to hescs (?), genetic memory, incomplete differentiation, possibility of teratoma obtaining tissue, viability of tissue, generation of sufficient number of cells, allogeneic may not be able to generate enough cells needed for therapy, may take too long to generate cells
20 Hurdles Scientific mechanisms to faithfully differentiate cells into desired cell types, scale-up while maintaining biological properties Clinical rigorously designed studies to evaluate not only efficacy, but also improvement over current therapies in order to bring about evidence-based changes in clinical practice Financial how will the health care industry deal with the cost of cell-based therapies Availability in the current climate, how will these new therapies be disseminated to all that need them
21 Acknowledgements Alexander Friedenstein* Sergei Kuznetsov Natasha Cherman Matt Phillips Paul Krebsbach* Mahesh Mankani* Arun Balakumaran* *past members Paolo Bianco* CPS/DTM/CC David Stroncek Marianna Sabatino SCU/NINDS Barbara Mallon Kye-Yoon Park Kevin Chen
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