stem cells, regenerative medicine and cgmp

Regenerative Medicine and Stem Cell Therapies

Regenerative Medicine

Major component of successful regenerated / tissue engineered organs– Scaffolds

• A critical element is the binding of the repopulating cells to a skeleton that approximates or is exactly like the organ being replaced or repaired

• Many reports of delivered stem cells not staying anchored, migrating to new sites or simply not surviving

• Artificial vs natural materials– Early attempts often included the need to surgically remove the scaffold after cells /

tissue / organ became established


Scaffolds – Several different materials have been discovered / developed that

have the characteristic of “dissolving”– Decellularization (aka, recellularization)

• Typically uses sodium dodecyl sulfate (SDS) and PBS to remove cells• Leaves complex natural scaffold and extensive capillary system• The matrix releases chemical signals that lead to the repopulation by stem

cells and generation of a functional organ• Successful human trials

– Bladder– trachea


Scaffolds - decellularization• Successful animal trials

– Lungs– kidneys– Heart

• Tengion – trials in animals where a partially repopulated organ (e.g., kidney) is returned to the animal donor species where indoginous signals lead to growth of functional organ


Scaffolds – Decellularization– Some controversy re: immune response

• Inflammation• Macrophage• T-cell (TH1 Vs TH2) • both allogeneic and xenogeneic

– Considered a “steping stone” technology• Currently unable to create a totally artificial scaffold• Eventually, will be successful, removing the need for donor organs


Using Stem Cells– aka, tissue-engineering

Approaches– Seeding scaffolds to grow and differentiate into organs

• May or may not include other multi- and/or uni-potent cells• What are the required / desired signals for proper differentiation?


Approaches– Direct delivery

• Injection at the actual site of injury• Via the bloodstream

– Applications in pre- and clinical trial• Several heart conditions• Bone repair• diabetes

Stem cell types in therapies

Human embryoninc stem cells– Mostly used in research, little to none in clinical trials– More nebulous is the role of umbilical cord cells

Adult stem cells– 2 types, multipotent (aka, tissue specific)– Mesenchymal cells

• Sourced from bone marrow, adipose tissue, et al

Induced Pluripotent Stem Cells (iPSCs)

Adult Stem Cells

Tissue specific stem cells Difficult to find, isolate and expand Little investigation into the use of these cells for therapeutic use

Mesenchymal stem cells Easy to obtain

Adipose tissue has nominally the highest density From humans – liposuction waste

Easy to expand Basic stem cell culture techniques

Easy to manipulate

Mesenchymal Stem Cells Autologous and allogeneic

– Patient specific– Real possibilities for “production”

Therapies (tissue-engineering)– Cardiomyocyte replacement following myocardial Infarct (MI)– Bone - muscle– Cartilage - marrow stroma– Tendon - other connective tissue

Mesenchymal Stem Cells (MSC)

Secrete spectrum of bioactive molecules– Many are immunosuppressive => allogeneic treatments– Query – is it the MSC that is the agent of repair or the bioactive

molecules; can the cells be eliminated in favor of just the appropriate bioactive molecules?

Provide a regenerative microenvironment– for a variety of injured adult tissues to limit the area of damage

and to mount a self-regulated regenerative response

Can be delivered to sites of injury by the bloodstream, i.e., by injection

Induced Pluripotent Stem Cells (iPSC)

Theoretically, from any somatic cell by reprogramming– Yamanaka cocktail – Oct4, Klf4, SOX2, c-Myc (aka, OKSM)

High potential for– Proliferation– Self-renewal– Pluripotent differentiation

Patient-specific therapy– Avoid immune rejection or harsh immunosuppressive drugs

Fewer ethical issues


Possible to correct genetic defect– Genetically alter (“fix”) somatic cell

• CRISPR/CAS-9

– Reprogram into iPSC– Return “normal” cell to patient – Already done in rodents

• Model for sickle cell anemia• Generate dopaminergic neurons to mitigate Parkinson’s disease (rat

model)


Used to make some disease models– Often difficult to find in animal models or cell culture assays– Modified (CRISPR/ CAS-9) to generate cell-level models

• Cardiomyocytes with heart disease• Juvenile on-set type I diabetes• Parkinson’s disease• ALS • other


Many challenges still faced– OKSM reprogramming efficiency is low

– Some cell systems, 0.001% up to 1%– Practice and experimentation is improving this aspect

– When using multiple or even single carrier• Commonly use lentivirus, up to four• Random genome insertion• Increased risk that the insertion point is in a critical site

– In a gene– Causes oncogene activation


Many challenges still faced– Tumorogenicity is still a problem

• Some success in leaving out the c-Myc insert• C-Myc is a major source of the oncogenic activity

– There is no current practical strategy for• Consistant iPSC differentiation• Purification of desired differentiated cell product• Failure to meet clinical standards

– Recent study indicates that iPSCs may be more immunogenic than originally thought.

stem cells, regenerative medicine and cgmp

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