Adult telomerase positive stem cells: Effects of biological agents on single cell clones of adult cells

 Corresponding author: Henry E. Young Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Adult telomerase positive stem cells: Effects of biological agents on single cell clones of adult cells Henry E. Young 1, 2, 3, 4, 5, 6, 7, * 1 Dragonfly Foundation for Research and Development, Macon, GA 31210 USA. 2 Henry E Young PHD Regeneration Technologies, Macon, GA 31210 USA. 3 Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA, 31210, USA. 4 Department of Surgery, Mercer University School of Medicine, Macon, GA, 31210, USA. 5 Department of Paediatrics, Mercer University School of Medicine, Macon, GA, 31210, USA. 6 Department of Obstetrics and Gynaecology, Mercer University School of Medicine, Macon, GA, 31210, USA. 7 Department of Anaesthesiology, Mercer University School of Medicine, Macon, GA, 31210, USA. GSC Advanced Research and Reviews, 2025, 25(03), 028-047 Publication history: Received 23 October 2025; revised on 30 November 2025; accepted on 02 December 2025 Article DOI: https://doi.org/10.30574/gscarr.2025.25.3.0369 Abstract The adult human body is composed of trillions and trillions of cells. These cells can be divided into three categories: telomerase negative differentiated cells, telomerase negative progenitor cells, and telomerase positive stem cells (aTPSCs). The aTPSCs are uniquely different from differentiated cells and progenitor cells, due mainly to not conforming to standard tissue culture practices, their limited number in the body, and the presence of the telomerase enzyme. Therefore, special technologies were developed to examine aTPSCs in vitro, which have been extensively outlined in recent publications. The aTPSCs and a tripotent progenitor cell, e.g., mesenchymal stem cell (MSC), were cloned from single cells using repetitive single cell clonogenic analysis with 5-6x exosome-conditioned medium. Within the body, both aTPSCs and progenitor cells, including MSCs, respond to environmental cues (biological activities) dictating their ability to repair/regenerate damaged cells and tissues. Four biological activities were noted during the repair/regeneration process, e.g., proliferation, progression, induction, and anti-differentiation. This study was designed to test these four biological activities on the single cell generated clones to determine their response. We utilized human recombinant proteins, morphogenetic proteins isolated from both demineralized bone matrix and sera, and cell-specific exosome conditioned media to test the hypothesis that aTPSCs and MSCs react both similarly and differently to biological activities inherent in the body, e.g., proliferation, progression, induction, and antidifferentiation. Keywords: Stem Cells; ATPSCS; MSCS; Proliferation; Progression; Induction; Anti-Differentiation 1. Introduction The adult human body is composed of trillions and trillions of cells. These cells can be divided into three categories: telomerase negative differentiated cells, telomerase negative progenitor cells, and telomerase positive stem cells (aTPSCs) [1]. Both progenitor cells and aTPSCs are involved in restoring and/or regenerating worn-out or damaged cells and tissues [2]. To do these activities, they respond to local environmental cues (exosomes) emanating from adjacent undamaged cells and tissues [3]. Four categories of biological agents have been identified, e.g., proliferative, progressive, inductive, and antidifferentiative. Various biological factors were utilized in this study, e.g., recombinant proteins, morphogenetic proteins GSC Advanced Research and Reviews, 2025, 25(03), 028-047 29 isolated from both demineralized bone matrix and sera, and cell-specific exosome conditioned medium. The recombinant proteins utilized were acidic-Fibroblast Growth Factor (a-FGF) [4-7], endothelial cell growth factor (ECGF) [8,9], vascular endothelial cell growth factor (VEGF) [10,11], transforming growth factor-beta (TGF-b) [12,13], basicfibroblast growth factor (b-FGF) [14,15,], erythropoietin (EPO) [16-18], c-Kit [19,20], Interleukin-6 (IL-6) [21,22], Nerve Growth Factor (NGF) [23,24], and Hepatocyte Growth Factor (HGF) [25,26]. The morphogenetic proteins utilized were bone morphogenetic protein-2 (BMP-2) [27,28] and bone morphogenetic protein-4 (BMP-4) [29,30]; as well as additional morphogenetic proteins isolated from demineralized bone matrix, e.g., skeletal muscle morphogenetic protein (SkMMP) [2], smooth muscle morphogenetic protein (SmMMP) [2], cardiac muscle morphogenetic protein (CdMMP) [2], adipocyte morphogenetic protein (AMP) [2], fibroblast morphogenetic protein (FMP) [2], scar fibroblast morphogenetic protein (ScFMP), tendon morphogenetic protein (TenMP), and ligament morphogenetic protein (LigMP) [2]; and morphogenetic protein-activities isolated from different lots of serum, e.g., blood vessel morphogenetic protein (BVMP) [31], brain morphogenetic protein (BrnMP) [31], liver morphogenetic protein (LivMP) [31], lung morphogenetic protein (LngMP) [31], pancreas morphogenetic protein (PanMP) [31], and keratinocyte morphogenetic protein (KerMP) [31]. Cell-specific exosome conditioned medium was derived from explant cultures of the following tissues/organs [3] and designated as “organ” conditioned medium (“X”CM), e.g., skeletal muscle conditioned medium (SkMCM), smooth muscle conditioned medium (SmMCM), cardiac muscle conditioned medium (CdMCM), tendon conditioned medium (TCM), ligament conditioned medium (LCM), bone (osteogenic) conditioned medium (OCM), cartilage conditioned medium (CCM), adipocytes conditioned medium (ACM), blood vessel conditioned medium (BVCM), fibroblast/fibrocytes conditioned medium (FCM), brain conditioned medium (BrnCM), liver conditioned medium (LivCM), lung conditioned medium (LngCM), pancreas conditioned medium (PanCM), keratinocyte conditioned medium (KerCM), Sertoli cells conditioned medium (SertoliCellCM). These biological agents were used to ascertain the response of clonal populations of aTPSCs and the tripotent MSCs to these compounds using the ELICA high throughput screening assay [32]. The hypothesis tested was that aTPSCs and MSCs react both similarly and differently to biological agents with their own unique activities inherent in the body, e.g., proliferation, progression, induction, and antidifferentiation. 2. Materials And Methods 2.1. Assay Reagents: [2,31-33] 2.1.1. Species-specific buffers • Amphibians and Reptiles – 10% Holtfreter’s solution [34] • Avians – Tyrode’s balanced salt solution, #T-2145 (Sigma) • Non-human mammals – Phosphate Buffered Saline, PBS (Sigma) • Humans – Dulbecco’s Phosphate-Buffered Saline (10X) #310-4080AJ (GIBCO) 2.2. Biological Agents 2.2.1. Anti-differentiative • Leukemia Inhibitory Factor (LIF) – R&D Systems • Anti-Differentiation Factor (ADF) – DFRD • Caffeine – Fisher Scientific • Scar Inhibitory Factor (SIF) – DFRD 2.2.2. Proliferative • Platelet-derived Growth Factor-AA (PDGF-AA) – R&D Systems • Platelet-derived Growth Factor-AB (PDGF-AB) – R&D Systems • Platelet-derived Growth Factor-BB (PDGF-BB) – R&D Systems 2.2.3. Progression • Insulin-Like Growth Factor-1 (IGF-1) – Cell Sciences • Insulin-Like Growth Factor-2 (IGF-2) – Cell Sciences • Insulin (Ins) - Sigma GSC Advanced Research and Reviews, 2025, 25(03), 028-047 30 2.2.4. Inductive • Bone morphogenetic protein-2 (BMP-2) – Genetics Institute • Osteogenic morphogenetic protein (OMP) – Isolated from demineralized bone matrix • Osteogenic conditioned medium (OCM) – generated from bone explant cultures • Cartilage morphogenetic protein (CMP) – Isolated from demineralized bone matrix • Cartilage conditioned medium (CCM) generated from cartilage explant cultures • Adipocyte morphogenetic protein (AMP) – isolated from demineralized bone matrix • Adipocyte conditioned medium (ACM) generated from Adipoblast/adipocyte explant cultures • Bone morphogenetic protein-4 (BMP-4) – Genetics Institute, • Acidic fibroblast growth factor (a-FGF) – R&D Systems • Endothelial cell growth factor (ECGF) – Sigma • Vascular endothelial growth factor (VEGF) – Genentech • Blood Vessel Morphogenetic protein (BVMP) – Isolated from serum • Blood Vessel Conditioned Medium (BVCM) generated from blood vessel explant cultures • Fibroblast morphogenetic protein (FMP) – Isolated from demineralized bone matrix • Fibroblast Conditioned Medium (FCM) generated from fibroblast/fibrocyte explant cultures • Scar Fibroblast morphogenetic protein (ScFMP) – Isolated from serum • Transforming growth factor beta (TGF-b) – Genentech • Basic fibroblast growth factor (b-FGF) – R&D Systems • Erythropoietin (EPO) – Amgen • c-Kit (c-Kit) – R&D Systems • Interleukin-6 (IL-6) – R&D Systems • Nerve growth factor (NGF) – Genentech • Brain Morphogenetic Protein (BrnMP) – Isolated from serum • Brain Conditioned Medium (BrnCM) generated from brain explant cultures • Hepatocyte growth factor (HGF) – Genentech • Liver Morphogenetic Protein (LivMP) – Isolated from serum • Liver Conditioned Medium (LivCM) generated from liver explant cultures • Lung Morphogenetic Protein (LngMP) – Isolated from serum • Lung Conditioned Medium (LngCM) - generated from lung explant cultures • Pancreatic Morphogenetic Protein (PanMP) – Isolated from serum • Pancreatic Conditioned Medium (PanCM) generated from pancreas explant cultures • Keratinocyte Morphogenetic Protein (KerMP) – Isolated from serum • Keratinocyte Conditioned Medium (KerCM) – generated from keratinocyte explant cultures • Sertoli Cell Conditioned Medium (SertoliCellCM) – generated from Sertoli cell explant cultures • Skeletal Muscle Morphogenetic Protein (SkMMP) – Isolated from demineralized bone matrix • Skeletal Muscle Conditioned Medium (SkMCM) generated from skeletal muscle explant cultures • Smooth muscle morphogenetic protein (SmMMP) – Isolated from demineralized bone matrix • Smooth muscle Conditioned Medium (SmMCM) generated from smooth muscle explant cultures • Cardiac muscle morphogenetic protein (CdMMP) – Isolated from demineralized bone matrix • Cardiac muscle Conditioned Medium (CdMCM) generated from cardiac muscle explant cultures • Tendon morphogenetic protein (TenMP) – Isolated from demineralized bone matrix • Tendon Conditioned Medium (TenCM) generated from tendon explant cultures • Ligament morphogenetic protein (LigMP) – Isolated from demineralized bine matrix • Ligament Conditioned Medium (LigCM) generated from ligament explant cultures 2.3. ELICA Fixative Reagents • Species-Specific Buffer • Paraformaldehyde, #P6146, Sigma • Glutaraldehyde, #G5882, Sigma • Sodium Azide, #S2002, Sigma • D-Glucose, # G-6138, Sigma 2.4. Inhibition and Exhaustion of Endogenous Peroxidases • 5% Sodium Azide, #S2002 Sigma GSC Advanced Research and Reviews, 2025, 25(03), 028-047 31 • 30% Hydrogen Peroxide, #H-1009, Sigma 2.5. Blocking Agents for ELICA • Horse serum, #H7889, Sigma • Goat serum, #200-6210-AG, DSHB • Porcine Serum, #P9783 Sigma • Bovine serum albumin, #A-7906, Sigma • Neonate Bovine Serum, #2002, BioCell • Fetal Calf Serum, #F0392, Sigma • Human Serum, #H4522, Sigma • Tween 20 (polyoxyethylenesorbitan), ChemPure, Curtain Matheson Scientific, Houston, TX • Nonidet P-40, P-40, #56007, BDH • Triton-X-100, #T-6878 (Sigma) • Powdered non-fat dry milk, Kroger’s • Gelatin (cold water fish soluble collagen), #935425, Sigma 2.6. Positive Standards • Calf thymus DNA type-I, #D-1501, Sigma • Myosin from chicken muscle, #M-7266, Sigma • Rabbit anti-myosin (skeletal-smooth), #M-7648, Sigma 2.7. Primary Probes • 12/101, Developmental Studies Hybridoma Bank (DSHB) • 31-2, DSHB • 5D2-27, DSHB • MF-1, DSHB • MF-5, DSHB • C3/1, DSHB • M3F7, DSHB • 22/18, DSHB • ALD-58, DSHB • MF-20, DSHB • B3/D6, DSHB • M-38, DSHB • D76, DSHB • ALD-66, DSHB • JLA-20, DSHB • SP1.D8, DSHB • CHI, DSHB • 5C6, DSHB • 33-2, DSHB • 2E8, DSHB • D3, DSHB • ID4B, DSHB • MF-30, DSHB • ABL-93, DSHB • 5-D-4, #69-625, ICN • Anti-myosin, #65-790-1, ICN • Anti-desmin, #65-793-1, ICN • Anti-vimentin, #69-127-2, ICN • Anti-type IV collagen, #69-108-1, ICN • Anti-type II collagen, #1320-01, Southern Biotechnology • Anti-heparan sulfate proteoglycan, #MAB459, Chemicon • CEA-CAM-1, DH (D. Hixson, Providence, RI) • HCEA, RB GSC Advanced Research and Reviews, 2025, 25(03), 028-047 32 • CEA, #111518, Sigma • CD66e, BD (Beckton-Dickinson) • Dh-TuAg1, DH • MC-480 (SSEA-1), DSHB • MC-631 (SSEA-3), DSHB • MC-813 (SSEA-4), DSHB • CD10, BD • CD90, BD • Thy-1, DSHB • CD56, BD • Pax-6, DSHB • FORSE-1, DSHB • Vimentin, DSHB • Nestin, DSHB • R401, DSHB • HNES, R&D • MAB353, Sigma • RT-97, DSHB • NF68, Prospec • S-100, DSHB • NF-145, R&D • N-200, R&D • 8A2, DSHB • NG2, DSHB • TH, DSHB • SV2, DSHB • DOPA, Thermo-Fisher • T8660, Sigma • Tuj1, DSHB • GFAP, • CNPase, Abcam • Rip, DSHB • MOSP, Sigma • MAB, DSHB • 40E-C, DSHB • VM-1, DSHB • CD13, BD, • OP-137, Antibodies Inc • F5D, DSHB • MF-20, DSHB • ALD-58, DSHB • A4.74, DSHB • IA4, Thermo-Fisher • Calp, Abcam • MAB-3252, • MAB1548, • WV1D1, • MP111, • CIIC1, DSHB • II-4CII, DSHB • HC-II, Abcam • D1-9, DSHB • 9/30, DSHB • 12/21, DSHB • 12C5, DSHB • H-CD34, BD GSC Advanced Research and Reviews, 2025, 25(03), 028-047 33 • CD31, BD • P1H12, DSHB • P2B1, DSHB • P2H3, DSHB • H-endo, Fisher Scientific • H5A4, DSHB • H4C4, DSHB • Hermes-1, DSHB • CD45, BD • CD63, BD • CD95, BD • H5A5, DSHB • H5C6, DSHB • HFSP, DSHB • 1B10, DSHB • H-AFP, R&D Systems • R-AFP, Abcam • DESMO, Abcam • LAP, DSHB • 151-Ig, DSHB • HA4c19, DSHB • OC2, DH • OC3, DH • OC4, DH • OC5, DH • OC10, DH • H.4, DH • H.1, DH • DPPIV, DH • OV6, DH • HESA, DSHB • YM-PS087, Sigma • INS, Sigma • YM-PS5088, Sigma • SOMA, Sigma • 11180, Sigma • Telom, Abcam • PI, Abcam • DAPI, Abcam • Gal-19, Abcam 2.8. Histochemical Stains • Von Kossa, Chroma-Gesellschaft • AlkPhos, Sigma • Mallory Heidenhain One-Step reagents, Chroma-Gesellschaft • Alcian blue, Chroma-Gesellschaft • pH 1.0 • pH 2.5 • Alcec blue, Chroma-Gesellschaft • pH 1.0 • 2.5 • Safranin-O, Chroma-Gesellschaft • pH 1.0 • pH 2.5 • Sudan Black-B, 199664, Sigma • Oil Red-O, 00625, Sigma GSC Advanced Research and Reviews, 2025, 25(03), 028-047 34 2.9. Verification of Histochemical Staining • Toluene, removes hydrocarbons from fat cells, Sigma • Hyaluronidase, Sigma • Chondroitinase-AC, Sigma • Chondroitinase-ABC, Sigma • Keratanase, Sigma • Heparanase, Sigma • EDTA, divalent cations (Ca+2, Mg+2, Zn+2, Ba+2), Sigma • EGTA, univalent cation (Ca+2 only), Sigma 2.10. Secondary Probes • Goat anti-rat osteocalcin, #BT-413, Biomedical Technology • Biotin, goat anti-mouse IgG, #OB1126-21, Fisher • Biotin, goat anti-mouse IgG (fab specific), #B-7151, Sigma • Biotin, goat anti-rabbit IgG, #OB1316-21, Sigma • Biotin-SP, goat anti-mouse IgG, #JGM-065003, Accurate • Biotin-SP, goat anti-mouse fab-specific, #JGM-066003, Accurate • Biotinylated affinity purified, rat adsorbed anti-mouse IgG (H + L) (BA-2001, Vector Laboratories) 2.11. Tertiary Probes • Streptavidin-peroxidase, #JSA-030084, Accurate • Avidin-peroxidase, #JSA-030083, Accurate • Monoclonal anti-goat IgG clone GT-34 biotin, #B-3148, Sigma • Horseradish peroxidase conjugated Avidin-D, #A-2004, Vector • Peroxidase Standard PK-4000 Vecstatin ABC Reagent Kit, Vector Laboratories 2.12. Reagents to Quantify or Visualize Tertiary Probes • ABTS kit, #506201, Kirkegaard and Perry • 4-Cloro-1-napthol, #C-8890, Sigma • 3,5-Diaminobenzoic acid dihydrochloride, #11-383-2, Aldrich • Vastatin ABC Reagent Kit, Vector Laboratories Inc., Burlingame, CA 2.13. Miscellaneous Supplies • Aqua-Mount (mounting coverslips), Vector Laboratories • Parafilm, #13-374-12, Dagger • Ethanol, Sigma • Isopropyl alcohol, Sigma • Micropipettors, Diagger • Nitrile Gloves, Dagger • Pipets: 1-ml, 2-ml, 5-ml, 10-ml, Diagger • 96-well plates, Costar/Corning 2.14. Equipment • Hera-2 Incubator, Thermo-Fisher • 96-well plate reader, R&D • Vortex Mixer, Thermo-Fisher • -70oC Ultra-Low Freezer, Thermo-Fisher • -80oC Ultra-Low Freezer, Thermo-Fisher • Liquid Nitrogen Dewer, Thermo-Fisher 2.15. Suppliers • Abcam, Cambridge, MA • Accurate, Accurate Chemical and Scientific Corporation, Westbury, NY GSC Advanced Research and Reviews, 2025, 25(03), 028-047 35 • AirGas, AirGas, Local Sore • Aldrich, Aldrich Chemical Co, Milwaukee, WI • Aldrich, Aldrich Chemical Co., Sigma-Aldrich, St. Louis, MO • Antibodies Inc, Davis, CA • Atlas, Atlas Biologicals, Fort Collins, CO • BDH, BDH Chemicals Ltd, Poole, England • BTH, Biomedical Technology Incorporated, Cambridge, MA • Chem, Chemicon, El Segundo, CA • ChemPure, Curtain Matheson Scientific, Houston, TX • Chroma-Gesellschaft, Roboz Surgical Co, Washington, DC • Costar/Corning, Life Sciences Div, Kennebunk, ME • Diagger Scientific, Vernon Hills, IL • DFRD, Dragonfly Foundation for Research and Development, Macon, GA • DH, Douglas Hixson, Department of Medicine, Brown University, Providence, RI • DSHB, Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained at the University of Iowa, Department of Biological Sciences, Iowa City, IA • Fisher-Biotech, Waltham, MA • Fisher, Fisher Scientific Co., Norcross, GA • GIBCO, GIBCO, Grand Island, NY • ICN, ICN Biomedicals, Costa Mesa, CA • Jackson, Jackson Laboratories, Bar Harbor, ME • Kodak, Kodak, Rochester, NY • KP, Kirkegaard and Perry Laboratories, Gaithersburg, MD • Kroger’s, Kroger’s, Macon, GA • Morton Thiokol, Morton International, Rohm and Haas, Philadelphia, PA • Prospec, Prospec, Rehovot, Israel • RB, Ray Biotech, Atlanta, GA • R&D Systems, Minneapolis, MI • SBA, Southern Biotechnology Associates, Birmingham, AL • Sigma, Sigma Chemical Co., St Louis, MO • Thermo-Fisher Scientific, Waltham, MA • Vector, Vector Laboratories, Burlingame, CA 3. Procedure Clonal populations of adult telomerase positive stem cells, e.g., totipotent stem cells (TSCs), halo-like stem cells (HLSCs), corona-like stem cells (CLSCs), pluripotent stem cells (PSCs), germ layer lineage stem cells (GLSCs), ectodermal stem cells (EctoSCs), mesodermal stem cells (MesoSCs), and endodermal stem cells (EndoSCs), and a clone of telomerase negative tripotent progenitor cell, MSCs, were incubated with physiological to pharmaceutical concentrations of biological agents to determine their response. Four categories of biological agents were identified: anti-differentiating agents, proliferative agents, progression agents, and inducing agents. Ninety-six well plates were utilized to devise a high throughput screening assay for the testing system [32]. One thousand cells were plated per well on a 1% type-1 collagen substratum for the aTPSCs and on uncoated plastic for the MSCs [35]. The cells were washed with incomplete culture medium (e.g., OptiMem + GlutaMax, 1% antibiotic/antimycotic, pH 7.4 [35], and then incubated with particular agents from physiological to pharmaceutical doses, e.g., nanogram to microgram quantities per ml, in complete medium containing 10% heat inactivated serum. Medium was changed, dependent on the color of the medium [3]. We utilized 130 immunocytochemical and histochemical staining procedures for specific phenotypic expression markers (Table 1), using an enzyme-linked immuno-culture assay (ELICA), to screen aTPSCs and MSC with biological agents to determine their response to these agents [32]. Table 1 Antibodies, Immunocytochemistry, and Histochemistry for Phenotypic Expression Markers Antibody Antigen Embryological Origin CEA-CAM-1 Carcinoembryonic antigen-cell adhesion molecule-1 Totipotent HCEA Human Carcinoembryonic antigen Totipotent GSC Advanced Research and Reviews, 2025, 25(03), 028-047 36 CEA Carcinoembryonic antigen Totipotent CD66e Carcinoembryonic antigen Totipotent DH-TuAg1 Spermatogonia Male Gamete MC-480 SSEA-1 Pluripotent MC-631 SSEA-3 Pluripotent MC-813 SSEA-4 Pluripotent CD10 Neutral endopeptidase Pluripotent AlkPhos Alkaline Phosphatase Pluripotent CD56 Neural cell adhesion molecule Ectoderm Pax-6 Neurogenic lineage Ectoderm FORSE-1 Neuronal precursor cells Ectoderm Vimentin Cells of neurogenic lineage Ectoderm Nestin Cells of neurogenic lineage Ectoderm R401 Nestin-neuronal lineage Ectoderm HNES Nestin-neuronal lineage Ectoderm MAB353 Nestin-neuronal lineage Ectoderm RT-97 Neurofilaments = neurons Ectoderm NF68 Neurofilament-68 = neurons Ectoderm S-100 Neurofilaments-100 = neurons Ectoderm NF-145 Neurofilaments-145 = neurons Ectoderm N-200 Neurofilaments-200 = neurons Ectoderm 8A2 Neurons Ectoderm NG2 Neurons Ectoderm TH Tyrosine hydroxylase, precursor to neural transmitters Ectoderm SV2 Synaptic vesicles Ectoderm DOPA Dopamine, transmitter of dopaminergic neurons Ectoderm T8660 Beta-tubulin-III Ectoderm Tuj1 Beta-tubulin Ectoderm GFAP Glial-fibrillary acidic protein Ectoderm CNPase Glial cells = oligodendrocytes and astrocytes Ectoderm Rip Oligodendrocytes Ectoderm MOSP Oligodendrocyte specific proteins Ectoderm MAB Oligodendrocyte marker Ectoderm 40E-C Radial cells and radial glial cells Ectoderm VM-1 Keratinocytes Ectoderm M3F7 Type-IV collagen, basement membrane Ectoderm and Mesoderm 31-2 Laminin, basement membrane Ectoderm and Mesoderm 5D2-27 Cell adhesion molecule Ectoderm and Mesoderm GSC Advanced Research and Reviews, 2025, 25(03), 028-047 43 Conditioned Medium, Pancreatic cells, e.g., pancreatic ductal cells, alpha-cells, beta-cells, delta-cells, PP-cells, epsiloncells; KerMP57, Ker Cells58, Keratinocytes; KerCM59, Ker Cells, Keratinocytes; Sertoli Cell-Conditioned Medium60, Spermaatogonia61, spermatogonia; SkMMP62, Skeletal Muscle Morphogenetic Protein, Skel Musc63, Skeletal Muscle; SkMCM64, Skeletal Muscle Conditioned Medium, Skel Musc, skeletal muscle; SmMMP65, Smooth Muscle Morphogenetic Protein, Smth Musc66, smooth muscle; SmMCM67, Smth Musc, smooth muscle; CdMMP68, Card Musc69, cardiac muscle; CbMCM70, Card Musc, cardiac muscle; TenMP71, Tendon Morphogenetic Protein; TenCM72, Tendon Conditioned Medium; LigMP73, Ligament Morphogenetic Protein, Lig74, ligament; LigCM75, Ligament Conditioned Medium, Lig, ligament. 5. Discussion In recent years, considerable attention has focused on the isolation and characterization of endogenous bioactive factors and their importance in influencing aspects of tissue development, maturation, aging, replacement, and repair. Demineralized bone matrix has been shown to contain a number of factors that influence proliferation, chemotaxis, angiogenesis, chondrogenesis, and osteogenesis [39-42]. Of particular interest has been the family of bone morphogenetic proteins (BMP) in the transforming growth factor–beta (TGF-β) superfamily [43-45], cartilage morphogenetic protein [46], TGF-β, basic fibroblast growth factor (b-FGF), insulin, insulin-like growth factor–1 (IGF-1), and insulin-like growth factor–2 (IGF-2) [40,47]. On the basis of the presence of these compounds in bone matrix, we fractionated a water-soluble extract from a demineralized bone matrix and assayed for additional bioactive activities. We discovered activity corresponding to multiple factors within the water-soluble fractions, e.g., skeletal muscle morphogenetic protein (SkMMP), smooth muscle morphogenetic protein (SmMMP), cardiac muscle morphogenetic protein (CdMMP), fibroblast morphogenetic protein (FMP), adipocyte morphogenetic protein (AMP), tendon morphogenetic protein (TenMP), ligament morphogenetic protein (LigMP), osteogenic morphogenetic protein (OMP), cartilage morphogenetic protein (CMP), and scar inhibitory factor (SIF), and their ability to alter structure and function of aTPSCs [48-53]. We postulated that the bioactive factors isolated from demineralized bone matrix—that is, BMPs, TGF-β, IGF-1, IGF-2, insulin, Sk-MMP, SIF, and so forth, were produced by cells not belonging to the osteogenic lineage. The cells producing these factors would presumably secrete the compounds into the blood, which would in turn be sequestered in the bone matrix. We therefore began analysing various lots of commercially available serum to determine if these serum lots contained other inductive activities. On the basis of their respective interaction with aTPSCs in vitro [2,51-53], we identified the following activities from serum and designated the compounds responsible for their activity as plateletderived growth factor (PDGF)-like (proliferative) activity, skeletal muscle morphogenetic protein (Sk-MMP), smooth muscle morphogenetic protein (Sm-MMP), adipocyte morphogenetic protein (AMP), fibroblast morphogenetic protein (FMP), SIF, Leukemia inhibitory factor (LIF)-like (inhibitory) activity, and antidifferentiation factor (ADF)- like (inhibitory) activity. Additional morphogenetic protein-like activity isolated from sera were blood vessel morphogenetic protein (BVMP), scar fibroblast morphogenetic protein (ScFMP), brain morphogenetic protein (BrnMP), liver morphogenetic protein (LivMP), lung morphogenetic protein (LngMP), pancreas morphogenetic protein (PanMP), keratinocyte morphogenetic protein (KerMP), cardiac morphogenetic protein (CdMMP), tendon morphogenetic protein (TenMP), and ligament morphogenetic protein (LigMP) [2,51-53]. In addition to biological agents identified from demineralized bone matrix and sera, we also generated cell-specific exosome-conditioned media utilizing cultured explants from the following tissues, e.g., osteoblasts/osteocytes, chondroblasts/chondrocytes, adipoblasts/adipocytes, blood vessels, fibroblasts/fibrocytes, brain, liver, lung, pancreas, keratinocytes, Sertoli cells, skeletal muscle myoblasts/myocytes, smooth muscle myoblasts/myocytes, cardiac muscle myoblasts/myocytes, tendon, and ligaments [3]. Utilizing these sources, four categories of biological agents were identified: Anti-Differentiating Agents, Proliferative Agents, Progression Agents, and Inducing Agents (Table 2). As shown, there were distinct similarities and differences with respect to clones of aTPSCs versus the clone of MSCs in their response to these biological agents (Fig. 1, Table 3). Table 3. Similarities and Differences between aTPSC clones and MSC clone. Anti-differentiative1, prevents differentiation of cells in the presence of inductive agents; Proliferative2, stimulates proliferation in cells, for aTPSCs that contain the telomerase enzyme, proliferation is essentially unlimited as long as the cells remain undifferentiated, once they begin to differentiate into progenitor cells, they lose the telomerase enzyme and assume the cell doubling number associated with particular species, e.g., for rodents = 6-8 population doublings [54] before cell senescence and cell death; Progressive3, accelerates the phenotypic expression of cell-committed progenitor cells, but has no response on stem cells; Inductive4, is dependent on subcategory of aTPSCs, TSCs will form all cells similar to day-four blastomeres, PSCs will form all cells similar to inner cell mass, EctoSCs will form all cells of the ectodermal lineage, MesoSCs will form all cells of mesodermal lineage, EndoSCs will form all cells of endodermal lineage, MSCs will only form bone, cartilage, and fat cells. GSC Advanced Research and Reviews, 2025, 25(03), 028-047 44 Table 3 Similarities and Differences between ATPSC clones and MSC clone Agent TSC PSC ECTOSC MESOSC ENDOSC MSC AntiDifferentiative1 +++ +++ +++ +++ +++ + Proliferative2 (Population Doublings) Unlimited Unlimited Unlimited Unlimited Unlimited R: 6-8 Progressive3 - - - - - +++ Inductive4 All Cells All Somatic Cells Only Ectodermal Lineage Cells Only Mesodermal Lineage Cells Only Endodermal Lineage Cells Only Bone Cartilage Fat 6. Conclusion The hypothesis tested was that ATPSCS and MSCs react both similarly and differently to biological activities inherent in the body, e.g., anti-differentiation, proliferation, progression, and induction. The results demonstrate that aTPSCs and MSCs reacted similarly with respect to anti-differentiative agents and proliferative agents, but differently with respect to progressive agents and inductive agents. Compliance with ethical standards Acknowledgments This work was supported by grants from Rubye Ryle Smith Charitable Trust, Dragonfly Foundation for Research and Development, MedCen Community Health Foundation, and MorphoGen Pharmaceuticals, Inc. I like to thank my longtime collaborator Dr. Asa C. Black Jr for his mentorship with respect to teaching and research; my clinical collaborators; research associates and assistants, e.g., J Floyd-Collins-Coleman, GF Long-Black, NL Henson, LW Blake, KC Hawkins, N Walsh, C Alena, MBL Cole, V Krishna, S Ellis, J Hudson, WJ Butler, PE Kross, J Wang, J Wong, D Hixson, and C Duplaa; my multitude of co-authors, clinical attendings, clinical residents, foreign and domestic graduate students, medical students, undergraduate students, and a high school student, for their insight, strong work ethic, and help in conducting this research. The antibody CEA-CAM-1 was generously provided by D. Hixson (Providence, RI). The antibody to SSEA4 was obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained at the University of Iowa, Department of Biological Sciences, Iowa City, IA. MC813 (SSEA-4) antibody was developed by D. Solter. Leukemia Inhibitory Factor (LIF), Platelet-derived Growth Factor-AA (PDGF-AA), Platelet-derived Growth Factor-AB (PDGF-AB), Platelet-derived Growth Factor-BB (PDGF-BB), Acidic fibroblast growth factor (a-FGF), Basic fibroblast growth factor (b-FGF), c-Kit (c-Kit), and Interleukin-6 (IL-6) were the kind gifts of R&D Systems. Insulin-Like Growth Factor-1 (IGF-1) and Insulin-Like Growth Factor-2 (IGF-2) were generously provided by Cell Sciences. Bone morphogenetic protein-2 (BMP-2) and Bone morphogenetic protein-4 (BMP-4) were the kind gifts of Genetics Institute. Vascular endothelial growth factor (VEGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and Transforming growth factor beta (TGF-b) were the kind gifts from Genentech. Disclosure of conflict of interest No conflict of interest was disclosed. Statement of ethical approval Animal Use The use of animals in this study complied with the guidelines of Mercer University’s Institutional Animal Care and Use Committee (ACUC). These guidelines reflect the criteria for humane animal care of the National Research Council as outlined in “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health GSC Advanced Research and Reviews, 2025, 25(03), 028-047 45 Statement of informed consent Human Use The use of human biopsy specimens in this study complied with the guidelines of Mercer University’s Institutional Review Board (IRB). 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