Regenerative medicine

Regenerative medicine encompasses medical treatments available and in research stages that aim to replace, regenerate, or restore function in human tissues and organs. Stem cell and tissue engineering technologies are used in regenerative medicine. Regenerative medicine treatments include cell-based therapy, biological or synthetic materials that promote tissue growth, and repair and scaffolds seeded with cells. Regeneration strategies to replace injured tissues with healthy tissues include the application of small molecule therapeutics that induce resident tissue to undergo reparative and regenerative processes and the application of biologics or chemicals to enhance cell-based therapies prepared ex vivo.

Regenerative medicine is considered a potential solution to challenges in organ transplantation, such as long waitlists for organ transplant surgeries and the side effects of immunosuppressant (antirejection) medications. Developmental biology, the study of the development of biological organisms from fertilized egg through differentiation into the various tissue types of an adult, overlaps with regenerative medicine since each strives to understand which molecular and physical cues are needed to specify different cell types.

Bone marrow transplants are a type of regenerative medicine that contains hematopoietic, or blood-forming, stem cells. Bone marrow treatment is not regulated by the FDA. Hematopoietic progenitor cells (HPCs) are descendants of stem cells and an intermediate cell type in blood cell development. HPCs develop into various cells of the blood and immune system. HPCs derived from umbilical cord blood were the only FDA-approved stem cell product as of 2019. Cancer immunotherapy, the activation, replacement, engineering, and regeneration of the immune system to fight cancer, is considered a form of regenerative medicine. Cancer immunotherapies such as CAR-T therapy, engineered T cells, and umbilical cord blood made up the majority of FDA-approved stem cell therapies as of 2022.

The following HPC transplantations are used to restore cells that constitute the blood and immune system due to inherited or acquired diseases or when cells of the blood and immune system need to be restored after myeloablative treatments, such as chemotherapy and radiation therapy.

• Allocord is a cord blood-derived HPC product manufactured at SSM Cardinal Glennon Children’s Medical Center

• Clevecord is a cord blood-derived HPC product manufactured at Cleveland Cord Blood Center

• Ducord is a cord blood-derived HPC product manufactured at Duke Medical School

• Hemacord is a cord blood-derived HPC product manufactured at New York Blood Center

• Cord blood-derived HPC product manufactured at Clinimmune Labs, University of Colorado Cord Blood Bank

• Cord blood-derived HPC product manufactured at MD Anderson Cord Blood Bank

• Cord blood-derived HPC product manufactured at LifeSouth Community Blood Centers, Gainesville, Florida

• Cord blood-derived HPC product manufactured at Bloodworks, Seattle, Washington

• Breyanzi (lisocabtagene maraleucel) is an autologous CAR-T cell therapy manufactured by Juno Therapeutics, a Bristol-Myers Squibb treatment for relapsed or refractory large B-cell lymphoma.

• Provenge (sipuleucel-T), manufactured by Dendreon Corp, is an autologous cellular immunotherapy for prostate cancer.

• Tecartus (axicabtagene cioleucel), manufactured by Kite Pharma, is a CAR-T therapy for treatment of adult patients with relapsed or refractory mantle cell lymphoma (r/r MCL).

• Yescarta (axicabtagene ciloleucel), manufactured by Kite Pharma, is a CAR-T therapy for large B-cell lymphoma.

Spinal muscular atrophy (SMA) is a childhood motor neuron disease, characterized by weakness and muscle wasting caused by a deficiency in survival motor neuron (SMN) protein. Without this protein, the motor neurons, the nerves that control muscle movement, are lost. Regenerative medicine therapies aim to enhance the ability of people with SMA to produce SMN protein.

• Zolgensma (onasemnogene abeparvovec-xioi), manufactured by Novartis Gene Therapies (previously AveXis), is a gene therapy that replaces the non-functional SMN1 gene with a functional copy of the gene.

• Spinraza (nusinersen), by Biogen, is an antisense oligonucleotide (ASO), which enhances the ability of a similar gene called SMN2 to produce full length SMN protein to compensate for the malfunctioning SMN1 gene which normally produces full length SMN protein. The ASO works by binding to the RNA transcript of the SMN2 gene and modifying RNA splicing.

• Risdiplam (Evrysdi), by Roche, is a small molecule product that modifies the splicing of SMN2 to increase production of full-length SMN protein.

Luxturna by Spark Therapeutics is a gene therapy product for the treatment of Leber congenital amaurosis, which is a type of retinal dystrophy. Luxturna delivers a functional copy of the RPE65 gene into retinal cells to restore vision.

MACI is a product comprised of autologous cultured chondrocytes on a porcine collagen membrane, manufactured by Vericel. The recipient’s own cartilage cells are used to produce this autologous cellularized scaffold product indicated for cartilage defects of the knee.

Gintuit is a product manufactured by Organogenesis, comprised of a biodegradable scaffold, bovine collagen, and human skin cells (keratinocytes and fibroblasts). The product is a cellular sheet applied topically to the gums and other oral tissue over surgically created vascular wound beds in the mouth. The mechanism of action is not fully identified, but in vitro studies in the laboratory have shown that Gintuit secretes factors known to promote wound repair and regeneration. Keratinocytes and fibroblasts are grown from precursor cells derived from the foreskin of circumcised babies.

LaViv(R) (Azficel-T), developed by Fibrocell Science, is used for wrinkles at the nasolabial folds, also known as smile lines. The recipient’s own skin cells are extracted and cultured to create the autologous cell therapy product, which is given by injection.

The term regenerative medicine began to be used in the 1990s. The idea of producing artificial organs was discussed earlier, by Alexis Carrell and Charles Lindbergh in their 1938 book, The Culture of New Organs. Bone marrow-derived hematopoietic (blood cell forming) stem cells have been used for treatment of blood disorders or after cancer treatments since the 1950s. The concept of tissue regeneration arises in Greek mythology in the story of the immortal Prometheus. In a punishment from Zeus, Prometheus was bound to a rock. An eagle was sent to feed on his liver each day, but his liver grew back each night.

Many organisms in nature are able to regenerate, including the limbs of the salamander. Humans can regenerate a severed fingertip until eleven years of age. In humans, skin stem cells are multipotent adult stem cells that can self-renew and differentiate into different cell lineages of the skin to participate in skin renewal and repair of injury.

Stem cells are unspecialized cells in the body that may be induced to differentiate into multiple different cell types. Stem cells have the ability to self-renew or self-replicate indefinitely. When a stem cell divides, one daughter cell may make a more specialized cell type while the other daughter cell remains a stem cell. Alternatively, stem cells may divide so both remain stem cells over multiple cycles to replenish the pool of stem cells.

Human embryonic stem cells (hESCs), derived from the inner cell mass of blastocyst-stage human embryos, were one of the first types of stem cells used in regenerative medicine. hESCs were first isolated in 1998 and received FDA approval for use in clinical trials to treat thoracic spinal cord injury and macular degeneration. Tumorigenic risk, immune rejection risk, and ethical dilemmas have limited the use of hESCs in regenerative medicine.

ESCs are pluripotent stem cells that retain the ability to form all three germ layers (endoderm, ectoderm, and mesoderm) but not extraembryonic structures such as the placenta. Germ layers are the primary cell layers with distinct characteristics, formed in the earliest stages of embryonic development. Endoderm is the innermost layer and develops into internal linings of the body, such as the gastrointestinal tract, the lungs, and the liver. Ectoderm forms the outer layer of the body, such as the epidermis (skin), mammary glands, and the central and peripheral nervous systems. Mesoderm cells originate between endoderm and ectoderm and give rise to the dermis of the skin, heart, muscle, bones, and the urogenital system.

Induced pluripotent stem cells (iPSCs) are a type of stem cell made by reprogramming adult somatic cells with four key transcription factors: OCT4, SOX2, KLF4, and c-MYC, collectively referred to as OSKM. Somatic cells are cells that are not germ cells (sperm and egg cells). Transcription factors control the activity of genes. iPSC cells are similar to embryonic stem cells, and they can be induced to differentiate or become a more specialized cell type. Human iPSCs have been differentiated into neurons, pancreatic cells, osteogenic cells, hematopoietic cells, cardiac cells, adipocytes, vascular cells, endothelial cells, and hepatocytes.

Many methods of generating iPSCs are not appropriate for clinical applications because the transcription factors are introduced into cells by viral or non-viral vectors that integrate into active genes. This carries risk because exogenous genetic factors are introduced and because flanking genes may become activated, potentially causing cancer. Introduction of the c-MYC gene carries a risk because deregulated expression of c-MYC is associated with many human cancers. In addition, partially differentiated iPSCs that remain in the iPSC product may have teratoma-forming potential.

Non-integrating methods for generating iPSCs include viral methods, such as adenovirus. Non-viral methods, which include DNA plasmids; episomal vectors; minicircle vectors; and delivery of mRNA, microRNA, proteins, and exosomes have shown lower reprogramming efficiency compared with lentiviral vector-based reprogramming. Exosomes are nanovesicles that are secreted by cells and circulate in body fluids that contain mRNA, miRNA, and proteins. A piggyBac transposons system is also used for iPSCs, which allows the removal of inserted genes after the reprogramming step.

Mesenchymal stromal cells, also called mesenchymal stem cells, abbreviated MSC, are harvested from adult bone marrow, periosteum, periodontal ligament, or adipose tissue. MSCs are thought to exert their effects through the secretion of factors that stimulate repair. MSCs are activated in damaged tissue and secrete signaling factors that calm inflammation and growth factors that stimulate cells in the tissue to repair and regenerate.

Strategies to enhance the generation of functional tissue-specific cell types tor cell-based transplantation therapy include the application of chemicals or small molecules to ex vivo cells isolated from patients or healthy donors. Certain chemicals induce desired gene expression, cell fate, and cell function and may also be used in vivo as therapeutics to induce endogenous cells in the body to repair or regenerate tissue.

The reprogramming of somatic cells into iPSCs through the addition of the OSKM genes (OSKM-mediated reprogramming) can be enhanced or partially replaced by small molecules that can be used to activate the expression of repressed genes. Many small molecules modulate gene expression/repression by changing the DNA methylation, histone acetylation, and histone methylation. DNA methylation, histone acetylation, and histone methylation are part of the epigenetic control of gene expression. These epigenetic modifications are not changes to the genetic code but are chemical and structural modifications that impact the way genes are packaged into the DNA-protein complex called chromatin. These epigenetic modifications cause certain genes to be in open chromatin or closed chromatin conformations that enable or prevent the gene from being active. Although different cell types contain the same genes, epigenetic differences allow different cell types to activate different subsets of genes.

During cellular reprogramming, DNA methylation is lost from regulatory regions of pluripotency-related genes. Small molecules that inhibit DNA methyltransferases or that target DNA demethylation processes may facilitate cellular reprogramming. Enzymes that modify histones such as histone acetyltransferases, histone deacetylases, histone methyltransferases (HMTs) and histone lysine demethylases (KDMs) may also be targeted by small molecules to affect cellular reprogramming. Preclinical research has shown that modification of epigenetics with small molecules can increase the efficiency of OSKM-mediated reprogramming and in some cases replace the need for the introduction of some OSKM genes.

Certain small molecules target signaling pathways to reprogram the composition of genes that are in active transcription. Afferent endocrine, paracrine, and cytokine signaling molecules cause cells to respond through the activities of interdependent molecules that form signal transduction pathways. Modulation of signaling pathways affects the activity of downstream transcriptional effectors and is a potential mechanism for cell reprogramming. SBB431542 and RepSox are small-molecule inhibitors of the signaling molecule TGFβ, that were shown to enhance iPSC reprogramming and replace Sox2. Only Oct4 was needed for iPSC reprogramming when A 83-01, an inhibitor of TGF-βRI, ALK4 and ALK7, was used on mouse or human somatic cells. Retinoic acid (RA) is another signaling molecule that is a target for cell reprogramming.

During cellular reprogramming towards pluripotency, cellular metabolism switches from oxidative respiration toward glycolysis. Small molecules can be used to promote glycolytic metabolism.

Decellularization is a process by which cellular components of living tissue are removed, and the remaining acellular extracellular matrix (aECM) scaffold is used as a natural scaffold for tissue engineering. Chemical or physical methods are used to remove cellular components and the aECM scaffold has bio-inductive properties on cells that are cultured on it, such as chemotaxis, attachment, migration, proliferation, and function in cells. The native vasculature is preserved, which allows the engineered tissues to be perfused and withstand physiological blood pressures.

Synthetic and naturally derived polymers may be combined to produce scaffolds that are compatible with human physiology. Naturally derived polymers include alginate, gelatin, collagen, fibrin, and hyaluronic acid. Synthetic polymers include polyethylene glycol. As of 2020, the resolution limit of bioprinter technology is about 2 μm for acellular constructs and 50 μm for constructs that include encapsulated cells. In preclinical research using animal models, cartilage and bone tissues have been produced with 3D bioprinting. Vascular, neurological, and lymphatic networks or tissues more than 1 cm in thickness are difficult to produce with 3D bioprinting.