CIRM US Stem cell NIH NOVA International Society for Stem Cell Research
Aging and Brain Repair (USF Health)
Companies: Stem Genomics
GPT-4b micro is an AI model developed with Retro Biosciences, designed for longevity science and protein engineering, particularly re-engineering Yamanaka factors (transcription factors Oct4, Sox2, Klf4 and c-Myc)to reprogram cells into stem cells with dramatically improved efficiency (up to 50x) for regenerative medicine. It’s a smaller, focused version of GPT-4, trained on biological data (protein sequences, structures) to optimize biological processes, showing promise in accelerating anti-aging research by compressing decades of work into weeks
Introduction:
A stem cell is defined by its ability to undergo asymmetric division, whereby one daughter cell differentiates and the other daughter cell is still a stem cell. The stem cell status of an HSC depends on its niche, which includes bone marrow stromal cells, the extracellular matrix and the local levels of cytokines. (Risitano “Advances in understanding the pathogenesis of acquired aplastic anaemia” 2018)
Types of Stem Cells
Due to their pluripotency, stem cells show great promise for treatments of many human diseases. Their ability to differentiate into any cell type makes them a valuable resource for research and development of treatments for diseases. Stem cells fall into 4 basic types, with 2 different mechanisms of action. Stem cells can be either pluripotent or multipotent. The two key types currently be pursued are mesenchymal stem cells (MSCs) or induced pluripotent stem cells (IPSCs). MSCs are found in bone marrow, adipose tissue, umbilical cold blood or peripheral blood. These cells can become new bone or carliage, fat, muscle or pancreatic beta cells. These cells, like iPSCs, have differentiation potential, meaning that they can turn into any type of cell. IPSCs have various sources and can become any adult cell type.
The majority of approve stem cell therapies use mesenchymal stem cells. These stem cells can differentiate into cell types that form connective tissues such as bone and cartilage and can promote connective tissue formation and repair, and reduce inflammation. Stem cell therapies for teh treatment of the tissue damage caused by heart attacks are moving through clinical trials.
In one type of therpay, induced pluripotent stem cells derived form a patient can have defective genes restored, before reintroduction back into the patient. This is being considered for a number of genetic diseases that result in blindess.
Somatic Stem Cells:
The starlet sea anemone, Nematostella vectensis, is potentially immortal, presumably because it possesses somatic stem cells which gives it an unusually high degree of regenerative potential. These somatic stem cells were identified recently by University of Vienna scientists in Science Advances. Transgenic reporter genes and single cell transcriptomics was used to identify cell ppopulations expressing the germline-associated markers piwi1 and nanos2 in the soma and germline. These are highly conserved genes that are usually active in humans only in germline lineages. But int he starlet sea anemone, they are active in both germline and somatic lieages.
Induced Pluripotent Stem Cells (iPSCs):
–Introduction:
omatic cells can be reprogrammed by transferring their nuclear contents into oocytes or by fusion with ES cells, incicating that unfertilized egss and ES cells contain factors that can confer totipotency or pluripotency to somatic cells.
In 2006, Keyoto University’s Kazutoshi Takahashi, PhD and Shinya Yamanaka, MD, PhD, reported that they had used defined factors to reprogram differentiated cells to an embryonic like state. This work led to induced pluripotent stem cells (iPSCs) that made it much easier to conduct stem cell research.
iPSCs are obtained form adult donor somatic cells. The cells are reprogrammed to pluripotency using a combination of genetic elements delivered into the cells by viral or other means. IPSCs proliferate indefinetly and maintain the potential to differentiate to nearly any functional cell type in the body. Thus, the cells can be gene edited, expanded and characterized to create master cell banks that can be used for every prouct batch. Because of those attributes, IPSCs have become one of the preferred starting materials for cell theary development.
Scientists at Genentech are using iPSC derived NGN2 induced neurons (or iNeurons) are differentiated form human iPSCs with transcription factor Neurogenin-w (NGN2) to rapidly become neurons.
–Transcription Factors which are important:
Several transcription factors, including Oct3/4 and Nanog function in the maintenance of pluripotency in both early embryos and ES cells. Several genes that are frequently upregulated in tumors, such as Stat3, E-Ras and beta-catenin have been shown to contribute to the long-term maintenance of the ES cell phenotype and the rapid proliferation of ES cells in culture. c-Myc and Klf4 are essential factors in maintaining plripotency (Yamanaka, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” Cell 126, 663-676, August 25, 2006).
—-c-Myc protein: has many downstream targets that enhance prolfieration and transformation, many of which may have roles in the generation of iPS cells. Of note, it associates with histone acetyltrasnferase (HAT) complexes, including TRRAP, which is a core subunit of the TIP60 and GCN5 HAT complexes and p300. Within the mamalian genome there may be up to 25,000 c-Myc binding sites and many more than the predicted number of Oct3/4 and Sox2 binding sites. c-Myc protein may induce global histone acetylation, thus allowing Oct3/4 and Sox2 to bind to their specific target loci. (Yamanaka, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” Cell 126, 663-676, August 25, 2006).
Cord Blood Stem Cells: Placental tissue is discarded routinely. It is very devoid of infectious pathogens. Companies involved in cord blood stem cells include Americord
Embryonic stem cells: ESCs are pluripotent cells derived from early stage embryos that have the capacity to differentiate into nearly any cell type. The two most widely studies embryonic stem cell types are mouse enbroyonic stem cells (mESC) and human embryonic stem cells (hESC). Maintenance of the undifferentiated state and pluripotency in mESC requires the presence of mouse fibroblast feeder layers (mEFs) or activation of STAT3 with leukemia inhibitory factor (LIF). Likewise, hESC are typically cultured on mEFs or in media obtained from growth of fibroblasts.
Hematopoietic stem cells: Pluripotent stem cells in the bone marrow of mammals have the potential to give rise to different types of blood cells which circulate in the peripheral blood. The pluripotent stem cells differentiate into various cell lineaages through multiple maturatational stages, thereby giving rise to committed blood cell types. Hematopoietic stem cells can be enriched by a procedure developed by I.L. Weissman and colleagues. In this procedure, a bone marrow sample is reacted with fluorescent monoclonal antibodies specific for antigens expressed on mature red and white blood cells. The labeled cells are then removed by flow cytometry with a fluorescence activated cell sorter leaving a number of stem cells in the sample. To further enrich the stem cells they can be incubated with various antibodies raised against cells likley to represent early differentiation stages in hematopoiesis (for example raised against antigen stem-cell antigen 1 in mice or CD34 which is present on 1-3% of hematopoeitic cells that can reconstitute the entire hematopoietic system in humans.) CellPro Inc. has reported successfull enrichment of CD34 stem cells using affinity chromatography. Such CD34 stem cells can be enriched and then engineered to replace any defective genes and then reintroduced back into the body. If some of the engineered stem cells are pluripotent, then all blood cells originating from those cells should have the healthy gene. In theory, patients would only need a single injection of the stem cells whereas if mature cells were used, multiple injections would be required.
The bone marrow microenvironment (BM niche), including immune cells, endothelium and mesenchymal stromal cells (MSC), constitutes specilized niches that orghestrate HSPC differentiation and maintenance, bone homeostasis and immunity.
–Markers:
—–CD34: serves as a valuable tool for identifying and isolating cruial cell populations, Its discovery has played a pivotal role in advancing understanidng of hematopoiesis, improving characterizqtion and manipulation of HSCs. Fiedorowicz, “Analytical considerations for gene-modified hematopoietic stem and progenitor cell therapies” Part 1- In-process drug substances and drug products, Bioprocess Interantional, 22(5), May 2024).
–Genome-modified hematopoietic stem and progenitor cells (GM-HSPC):
GM-HSPC therapies represent a significant frontier in the realm of personalized medicine, holding the promise of targeted interventions for a spectrum of disorders far beyond hematological conditions. The journey toward gene edited stem cell therapies begain with the work of E. Donnall Thomas during the 1950s and 1960s which demonstrated regenerative potential of HSC grafts transplanted into recipients. The 1970s brought a shift toward allegeneic HSC transplants. During the 1980s significant strides were made in improved conditioning regimens, optimizing the success of HSC grafts. These regimens involved pretransplant chemotherapy or radiation to create a conductive envionment for donor cell engraftment, which helped improved patient outcomes. The understanding about CD34 as a cell surface antigen and marker for hematopoeitic stem and progentior states was a significant milestone. (Fiedorowicz, “Analytical considerations for gene-modified hematopoietic stem and progenitor cell therapies” Part 1- In-process drug substances and drug products, Bioprocess Interantional, 22(5), May 2024).
–Applications of CD34 stem cells:
After AMI, damaged tissue secretes a complex blend of cardioactive chemokines that recruits and stimulated CD34+ cells, when then travel form bone marrow to peripheral blood. Post AMI peripheral blood concentrations of cD34+ cells correlated significantly with heart regeneration and functional improvement. CD34+ cells promote cardiac repair by releasing soluble paracrine factors and exosomes containing microRNA 9miRNA) molecules that induce antiogenesis and revascularization of damaged tissue. Those paracrine factors also enhance proliferation of resident cardiomyocytes, reducing fibrosis and attentuating remodeling effects. Scar-assocaited chemokines attract CD34+ cell to the ischemic zone and induce their comittment into endothelial pathways. Cell Prothera, a French clinical stage biotechnology company develops cell based therapies for ischemic diseases and has demonstrated the feasibility of intramyocardial injection of cluster of differentiation 34 (CD34) positive stem cells in patients who experienced acute myocardial infarcion. (Garitaonandia “Regenerative Medicine for Cardiovascular Disease”” BioProcess International, 22(4), April 2024)
Liu, (“Cytokine polarized, alternatively activated bone marrow neutrophils drive axon regeenration” Research Square) describe novel populations of BM dervied myeloid cells possessing neuroprotective and pro-regenerative properties. These cells were generated in vitro via short term culture of human CD34+ BM stem cells, with a combination of recombinant IL-4 and G-CSF. The findings suggested that fundamental characteritics of IL-4/G-CSF polarized neuro-regenerative myeloid cells cells. Wehther o mouse or human origin, these polrized cells, responsible for triggering axon regrowth, have a cell surface phenotype and transcriptomic signature consistent with immature, alternatively activated neutrophils. Both the mouse and human myeloid subseets epxressed eGFR ligands (HB-EGF for mice and TGFalpha for human), along with a panoply of other neuroprotective agetns and grwoth factors, that oculd contribute to their reparative functions.
Hepatic stellate cells (HSC): are the major profibrogenic cells in the liver. During liver injury, HSC are activated to a myofibrolastic phenotype. Together with protal fibroblasts and septal myofibroblasts of bone marrow origin, activated HSC produce most of the collagen matrix in injured livers.
Mesenchymal stems cells (MSCs): MSCs are multipotent adult stem cells that have been found in several different tissues, including umbilical cord blood, amniotic fluid and adipose tissue, as well as bone marrow. MSCs can self renew and have the multi lineage potential of differentation into a variety of cell types, including osteoblasts, chondrocytes, myoctes and adipocytes and also several different types of stromal cells, incluidng muscle, cartilage, marrow stroma and fibroblasts. MSCs have also been reported to have another unique characteristic (tropism migration) which facilitates migration to damaged tissue sites, such as sites with inflammation or wounds and sites with cancer cells. Cho (Gynecologic Oncology, 123 (2011) 379-386. Only MSCs posses the immunobmodulatory ability and are well tolerated during allogenic transplantation.
Over 900 clinical trials involving MSCs are listed on ClinicalTrials.gov but no MSC based therapics have been aproved in the US. (Kimbrel “Next-generation stem cells — ushering in a new era of cell-based therapies” Nature Reviews: Drug Discovery, 19, July 2020).
–Orbital fat-dervied stem cells (OFSCs): are MSCs isolated form human orbital fat tissue. They lack immunogenecity, and the safety and immunomodulatory ability of systemic OFSC transplantation has been demonstrated. In addition, OFSCs possess the osteogenic, chondrogenic and adipogenic differentiation capacity , and may differentiate into cornal epithelial cells upon contact with human corneal epithelial cells (Lin, Stem cell Research & therapy 2013, 4 72)).
Mesenchymal stromal cells (MSCs): are multipotent, self-renewing progenitor cells that can differentiate into adipocytes, chondrocytes and osteocytes. Cultured MSCs are plastic-adherent and spingle-shaped, and they express cell-surface markers CD44, CD73, CD90 and CD105, but not CD14, CD34, CD45, CD11b, CD79a, CD19 or HLA-DR. First isolated from bone marrow, human MSCs have been investigated extensively in clinical studies. MSCs also have been isolated from adiope tissue and peripheral blood. Perinatal organs and tissues such as aminiotic membrane, placenta, and umbilical cord also have been shown to be rich source of MSCs. The ability of MSCs to secrete high levels of proangiogenic and antinflammatory cytokines has been shown to enhance wound healing. (Phan, “Deriving messenchymal stromal cells from umbilical cord lining and Wharton’s Jelly”, Bioprocess Technical Journal, 2022).
A formulation developed from skin regeneration technology, CALECIM® is a product that encourages skin to regain its youthfulness. CellResearch Corporation’s, CALECIM® Cosmeceuticals Pte Ltd, has formulated CALECIM® into a range of skin care products.
The International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell (ISCT MSC) committee states that the term mesenchymal stem cell is not equivalent or interchangeable with mesenchymal stromal cell (MSC). The former refers to a stem cell population with demonstrable progenitor cell functionality of self-renewal and differentiation, whereas the latter refers to a bulk population with notable secretory, immunomodulatory and homing properties. The ISCT’s MSC committee further issued a minimal criteria to define multipotent MSCs as being plastic adherent, expressing CD73, CD90 and CD105, lacking the expression of hematopoietic and endothelial markers CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR and capable of in vitro differentiation into adipocyte, chondrocyte and osteoblast lineages. (Sensebe, “Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell committee position statement on nomenclature” Cytotherapy, 2019).
MSCs have garnered extensive attentaion becasue of their unique properites, including their multilineage differentation and immunomodulatory effects. To date, 474 clinical trails ahve been completed using MSCs to treat an array of diseases, such as acute myocardial infarction, knew osteoarthritis, rhematoid arthritis and diabetes. Despire the widespread interest in MSCs, they are scarce in humans, constituting just 0.01% and 0.005% of the bone-marrow mononucleated cells population for males and femailes. With such low cell counts, large volumes of bone-marrow aspirate and extended culture times are needed for sufficient MSC expansion in downstream processing and therapeutic applciations.
Production of Stem Cells
Under normal conditions, stem cells are anchored to the bone marrow at least in part through a bond between a particular receptor (CXCR-4) located on the stem cell and a protien (SDF-1) produced in the bone marrow. Plerixafor releases the stem cells into the bloodstream by disrupting that bond.
US Patent No. 7,897,590 discloses a method to obtain progenitor and/or stem cells by adminsitering G-CSF to a subject, administering plerixafor or a pharmaceutically acceptable salt in an amount effective to mobilize the progenitor and/or stems cells and harvesting the progenitor and/or stem cells.