Adult Telomerase Positive Stem Cells: Isolation, Plating and Propagation

 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: Isolation, Plating and Propagation 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 Pediatrics, Mercer University School of Medicine, Macon, GA, 31210, USA. 6 Department of Obstetrics and Gynecology, Mercer University School of Medicine, Macon, GA, 31210, USA. 7 Department of Anesthesiology, Mercer University School of Medicine, Macon, GA, 31210, USA. GSC Advanced Research and Reviews, 2025, 25(02), 407-440 Publication history: Received 06 October 2025; revised on 20 November 2025; accepted on 22 November 2025 Article DOI: https://doi.org/10.30574/gscarr.2025.25.2.0355 Abstract The adult human body is composed of trillions and trillions of cells. These cells can be divided into three categories: functional [differentiated] cells comprising 40%, maintenance [progenitor] cells comprising 59%, and healing [stem] cells comprising 1% of the cells of the body. Healing stem cells were discovered in 1975 residing within the connective tissue stroma of adult terrestrial salamanders undergoing complete limb regeneration. They were shown to form all damaged or lost tissues of an amputated limb, thereby restoring function to the limb. Since 1975, these cells have been extensively characterized. The healing stem cells are uniquely different from functional cells and maintenance cells. Healing cells are telomerase positive which gives them an essentially unlimited proliferation potential. There are 8 distinct subcategories of healing stem cells based on multiple parameters, including differentiation potentials. In vivo, they do not synthesize and secrete their own substratum, and therefore one must be provided for them. They can form multiple cell layers in vitro. In situ, they reside in connective tissue niches interspersed throughout the body as dormant, hibernating, quiescent cells. They can be stimulated to proliferate and mobilize into the blood stream as non-activated cells, but they need to be activated to function to replace damaged cells and tissues. Activation occurs by unmasking homing receptors on their cell surfaces and unmasking cell surface receptors for exosomes containing biological agents. Due to their uniqueness, special methodologies were developed for their isolation, plating, and propagation. This report describes those methodologies in detail. Keywords: Differentiated Cells; Progenitor Cells; Stem Cells; Isolation; Propagation; Segregation; Telomerase Positive; Adult 1. Introduction The adult human body is composed of trillions and trillions of cells. These cells can be divided into three categories: functional differentiated cells, maintenance progenitor cells, and healing stem cells [1]. The 220+ functional differentiated cells comprise approximately 40% of the cell types in the body. Functional differentiated cells are composed of parenchyma (functioning cell) and stroma (it’s connective tissue stroma). Examples of functional differentiated cells are signal transmitting neurons [2,3], hormonal secreting pancreatic islet cells [4-6], ambulatory skeletal muscle cells [7-9], and detoxifying hepatocytes [10-12]. GSC Advanced Research and Reviews, 2025, 25(02), 407-440 408 Table 1 Attributes of Endogenous Adult Telomerase Positive Healing Stem Cells Attributes TSCs1 HLSCs2 CLSCs3 PSCs4 GLSCs5 EctoSCs6 MesoSCs7 EndoSCs8 Size, microns 0.1-2.0 >2-4 >4-<6 6-8 >8-<10 10-12 10-12 10-12 0.4% Trypan blue Entire Cell Positive9 Halo10 Positive, Negative Corona11, Positive Negative Entire Cell Negative 12 Entire Cell Negative Entire Cell Negative Entire Cell Negative Entire Cell Negative Animal Cell Surface Markers CEA-CAM-113 CEA-CAM-1high SSEA-4low CEA-CAM-1low SSEA-4high SSEA-414 SSEA-4, Thy-115 Thy-1 Thy-1 Thy-1 Human Cell Surface Markers CD66e16 CD66ehigh CD10low CD66elow CD10high CD1017 CD10 CD9018 CD56, CD90 MHC-119 CD13, CD90 MHC-1 ??? CD90 MHC-1 Expressed Genes Telom +Bcl-2+ Nanog + Nanos + CXCR4 + Telom +NYD Telom +NYD Telom + Oct-3/4 +SonicHedge Hog + Telom + NYD Telom +NYD Telom +NYD Telom + NYD Culture Conditions Substrate Adhesion Substrate Adhesion Substrate Adhesion Substrate Adhesion Substrate Adhesion Substrate Adhesion Substrate Adhesion Substrate Adhesion Differentiation Capabilities22 Somatic Cells, Gametes, NP of IVD Somatic Cells Only24 Somatic Cells only Somatic Cells only Somatic Cells only Ectoderm Lineage Only25 Mesoderm Lineage Only26 Endo-derm Lineage Only27 Maximum Proliferation To Date >300 Rat Population Doublings >300 Rat Population Doublings >300 Rat Population Doublings >400 Rat Population Doublings >400 Rat Population Doublings >400 Rat Population Doublings >690 Human Population Doublings >400 Rat Population Doublings Doubling Time 12-14 hours 13-15 Hours 13-15 hours 14-16 hours 16-18 hours 18-24 hours 18-24 hours 18-24 hours GSC Advanced Research and Reviews, 2025, 25(02), 407-440 409 Table 1. TSCs1, totipotent stem cells; HLSCs2, halo-like stem cells; CLSCs3, corona-like stem cells; PSCs4, GLSCs5, germ layer lineage stem cells; EctoSCs6, ectodermal stem cells; MesoSCs7, mesodermal stem cells; EndoSCs8, endodermal stem cells; entire cell demonstrating Positive9 staining for 0.4% Trypan blue; Halo10, complete peripheral rim of positive trypan blue staining with central area of cell negative for Trypan blue staining; Corona11, crown of positive Trypan blue staining, remainder of cell is negative for Trypan blue staining; entire cell demonstrating Negative13staining for 0.4% Trypan Blue; CEA-CAM-113, carcinoembryonic antigen-cell adhesion molecule-1; SSEA-414, stage-specific embryonic antigen-4; Thy-115, N-glycosylated glycophosphatidylinositol (=CD90); CD66e16, carcinoembryonic antigen; CD1017, common acute lymphoblastic leukemia antigen (CALLA); CD9018, N-glycosylated glycophosphatidylinositol (=Thy-1); MHC-119, self-recognition molecule Major Histocompatibility Complex-Class-1; Suspension20, cells grow in suspension culture only; Substrate Adhesion21, only grows attached to a substrate; Differentiation Capabilities22, differentiation capabilities; All Cells23, will form all somatic cells of the body, the gametes (spermatogonia and oogonia), and the nucleus pulposus of the intervertebral disc (the only tissue derived from the notochord in adults); Somatic Cells Only24, will only form somatic cells of the body, will not form gametes, will not form nucleus pulposus of the intervertebral disc; Ecto Lineage Only25, will only form cells of the ectodermal germ layer lineage, will NOT form cells of either the mesodermal or the endodermal germ layer linages; Meso Lineage Only26, will only form cells of the mesodermal germ layer lineage, will NOT form cells of either the ectodermal or the endodermal germ cell lineages; Endo Lineage Only27, will only form cells of the endodermal germ layer lineage, will NOT form cells of either to ectodermal or mesodermal germ layer lineages; NYD28 , not yet determined [13]. Reprinted with permission from Young HE, Speight MO. Characterization of endogenous telomerase-positive stem cells for regenerative medicine, a review. Stem Cell Regen Med 2020; 4(2):1-14. The 230+ maintenance progenitor cells compose approximately 59% of the cells of the body and are the immediate precursor cells to the functional differentiated cells. There are four subcategories of maintenance progenitor cells: multipotent, tripotent, bipotent, and unipotent [13]. An example of a multipotent maintenance progenitor cell is the “hematopoietic stem cell” [14-16]. It will form 18+ cell types all within the hematopoietic lineage. An example of a tripotent maintenance progenitor cell is the “mesenchymal stem cell” identified by Caplan [17]. It will form three cell types, e.g., fat, cartilage, and bone [13,17-19]. An example of a bipotent maintenance progenitor cell is the adipofibroblasts. It will form only two cell types: fat cells and fibroblasts/fibrocytes [20,21]. Examples of unipotent maintenance progenitor cells are osteoblasts forming osteocytes (bone), cardiac myoblasts forming cardiac myocytes, and fibroblasts forming fibrocytes [22]. Healing stem cells (e.g., adult telomerase positive stem cells, aTPSCs) comprise approximately 1% of the ~450+ cell types (~220+ differentiated functional cells and ~230+ maintenance progenitor cells) of the body. There are three major categories and eight subcategories of healing stem cells (aTPSCs). The first major category of aTPSCs are the totipotent stem cells (TSCs) which will form all somatic cells of the body, gender-specific gametes, placental cells (that synthesize and secrete chorionic gonadotrophin), and the nucleus pulposus of the intervertebral disc (the only adult derivative of the notochord). The second major category of aTPSCs are the pluripotent stem cells which will form all somatic cells of the body, but will not form gametes, placental cells, or the nucleus pulposis of the intervertebral disc. Pluripotent stem cells are composed of multiple populations of cells in transition, e.g., halo-like stem cells (HLSCs), transitioning to corona-like stem cells (CLSCs), transitioning to pluripotent stem cells (PSCs), and transitioning to germ layer lineage stem cells (GLSCs). The third major category of aTPSCs are the germ layer lineage ectodermal stem cells (EctoSCs) forming all somatic cell types within the ectodermal germ layer lineage; the germ layer lineage mesodermal stem cells (MesoSCs) forming all somatic cell types within the mesodermal germ layer lineages; and the germ layer lineage endodermal stem cells (EndoSCs) forming all somatic cell types within the ectodermal germ layer lineage, (Table 1, Figs. 1-3) [13,23]. GSC Advanced Research and Reviews, 2025, 25(02), 407-440 410 Figure 1 Diagrammatic representation of size comparison and visual staining patterns viewed by brightfield microscopy of aTPSCs (e.g., TSCs, HLSCs, CLSCs, PSCs, GLSCs, EctoSCs, MesoSCs, and EndoSCs) and Progenitor Mesenchymal Stem Cells (MSCs) when stained with either 0.4% Trypan Blue or Carcino-Embryonic Antigen-Cell Adhesion Molecule-1 (CEA-CAM-1) antibody [24]. Reprinted with permission from Young HE. Adult telomerase positive stem cells: introduction and location. GSC Advanced Research and Reviews. 2025; 25(02): 296-331. Figure 2 Identification of eight categories of aTPSCs and their unidirectional differentiation potentials into telomerase negative progenitor cells and telomerase negative differentiated cells using specific objective assays of cell surface markers and inherent phenotypic expression markers [13]. Reprinted with permission from Young HE, Speight MO. Characterization of endogenous telomerase-positive stem cells for regenerative medicine, a review. Stem Cell Regen Med 2020; 4(2):1-14 GSC Advanced Research and Reviews, 2025, 25(02), 407-440 411 Figure 3 Diagrammatic representation of unidirectional transitioning of aTPSCs into maintenance/progenitor cells and subsequently into functional/differentiated cells with respect to size, Trypan blue staining, cell surface markers, expressed genes, and growth in culture [13]. Reprinted with permission of Young HE, Speight MO. Characterization of endogenous telomerase-positive stem cells for regenerative medicine, a review. Stem Cell Regen Med 2020; 4(2):1-14. Healing stem cells were discovered in 1975 residing within the connective tissue stroma of adult terrestrial salamanders undergoing complete limb regeneration [25,26]. Healing stem cells were shown to form all damaged or lost tissues of an amputated limb, thereby restoring function to the limb [27-30]. Since 1975, healing stem cells have been extensively characterized utilizing multiple parameters [13]. They are composed of telomerase positive 0.1-2-micron cells totipotent stem cells (TSCs), >2.0 - <10.0-micron pluripotent stem cells (HLSCs, CLSCs, PSCs, and GLSCs), and 10-12micron germ layer lineage stem cells (EctoSCs, MesoSCs, and EndoSCs). The 0.1-2.0-micron TSCs and 6-8-micron PSCs are seen at the 8-cell stage embryo (Fig. 4), suggesting that they probably arise at the 4-cell stage embryo through asymmetrical division of the blastomeres [31,32]. Figure 4 Scanning electron micrograph of a male embryo, somewhere between 8-cell stage and morula. Note presence of at least three pairs of totipotent stem cells (TSCs) and at least one pluripotent stem cell (PSC). Size approximation same as seen with flow cytometry (TSCs are 0.1 to 2 microns and PSC are 6 to 8 microns in size) [31]. Reprinted with permission from Young HE, Black AC. Pluripotent Stem Cells, Endogenous versus Reprogrammed, a Review. MOJ Orthop Rheumatol. 2014; 1(4): 00019 GSC Advanced Research and Reviews, 2025, 25(02), 407-440 412 The aTPSCs, specifically PSCs and TSCs, and progenitor cell seen in a plated mixed cell population derived from fresh isolates using type-1 collagenase and dispase enzymatic digestion of solid tissues (Fig. 5). Figure 5 Fresh mixed isolate of aTPSCs and progenitor cells. Note presence of single PSC (large arrowhead), a portion of a progenitor cell (asterisk), and two TSCs (small arrow) in photograph. Of the two TSCs in the photograph one is in prophase (left) and the other (right) is in metaphase (see chromosomes along metaphase plate). Magnification of original photograph, 800x, electronically enlarged to 1600x [33]. Reprinted with permission from Young HE and Black Jr AC. Naturally occurring adult pluripotent stem cells. In: Stem Cells: From Biology to Therapy, Advances in Molecular Biology and Medicine. 1st Ed, R.A. Meyers, Ed, WILEY-BLACKWELL-VCH Verlag GmbH & Co. KGaA. Chap 3, pp. 63-93, 2013. [Labelling differences between original paper and above Figure 5. PSCs = ELSCs and TSCs = BLSCs] GSC Advanced Research and Reviews, 2025, 25(02), 407-440 413 Table 2 Comparison / Contrast of Functional Cells, Maintenance Cells, and Healing Cells Attributes Differentiated Cells Maintenance Cells Healing Cells Healing Cells Healing Cells EctoSCs, MesoSCs, EndoSCs PSCs TSCs % in adults 40 59 0.9 0.09 0.01 Telomerase Enzyme Absent Absent Present Present Present Location Throughout the body Adjacent to Differentiated Cells Connective Tissues (CT) Niches CT Niches CT Niches Age Range Newborn to Geriatric Newborn to Geriatric Newborn to Geriatric Embryonic to Geriatric Embryonic to Geriatric Numbers with Aging Decline with age Decline with age Remain Constant Remain Constant Remain Constant Native Naïve State NA Quiescent Quiescent Quiescent Quiescent Teratoma Formation Absent Absent Absent Absent Absent Growth In vitro Single Layer Confluent Single Layer Confluent Single Layer Confluent Multi-Layered Confluent Multi-Layered Confluent Respond to Inhibitory Factors Yes Yes Yes Yes Yes Respond to Proliferative Factors Yes Yes Yes Yes Yes Proliferation Potential 50-70 PDs in Humans 6-8 PDs Rodents 50-70 PDs in Humans 6-8 PDs Rodents Unlimited Humans & Rodents Unlimited Humans & Rodents Unlimited Humans & Rodents Respond to Progression Factors NA Yes No No No Respond to Inductive Factors No Only in Committed Lineage Only in Committed Lineages Yes Yes Cell Types Formed NA Only in Committed Lineage Ectodermal, Mesodermal, Endodermal All Somatic Cells All Somatic Cells, NP of IVD, Gametes, Placenta GSC Advanced Research and Reviews, 2025, 25(02), 407-440 414 Time Period Fresh Isolate to In Vivo Use 4 hours 4 hours 4 Hours 4 hours 4 hours Time Period Fresh Isolate to Ex Vivo Use 1-24 hours 6-8 hours 24 hours 24 hours 24 hours Treatment Number Potential Thousands Millions Billions Billions to Trillions Billions to Trillions Express Self-Recognition Molecules Yes Yes Yes No No Immuno-Protected No No No Yes Yes Autologous Treatments Yes Yes Yes Yes Yes Allogeneic Treatments No Induces GvHD1 No Induces GvHD No Induces GvHD Yes Yes GSC Advanced Research and Reviews, 2025, 25(02), 407-440 415 Table 2. Attributes of functional differentiated cells, maintenance progenitor cells, and healing stem cells. 1GvHD, Graft versus host disease. If recipient has competent immune system, donor cells will be destroyed. If recipient has compromised immune system, donor cells will try to kill the donor [13]. Reprinted with permission from Young HE, Speight MO. Characterization of endogenous telomerase-positive stem cells for regenerative medicine, a review. Stem Cell Regen Med 2020; 4(2):1-14. Due to their limited number in the body and their uniqueness in not conforming to standard tissue culture practices [34], special methodologies were developed to examine adult telomerase positive stem cells both in vitro and for their use in vivo. Previous reports have been published demonstrating their in vitro characterization [13,23,35] and in vivo use in both pre-clinical IACUC-approved animal models [35-40] and in IRB-approved compassionate use human clinical diseases [36,37,40-55]. This begins a series of articles, explaining in step-by-step detail, the methodologies (e.g., rationale, standardization, reagents, manufacturers, catalog numbers, step-by-step instructions, and examples of expected results via text, tables, and figures) that were developed for in vitro characterization of aTPSCs and their use in vivo. The current report describes the methodologies developed to optimize the viability of the aTPSCs during their isolation, plating, and propagation for in vitro analyses. The hypothesis tested was “methodologies could be developed to isolate, plate, and propagate adult telomerase positive stem cells”. 2. Materials and methods 2.1. Reagents: [13,23] 2.1.1. Species-specific buffers: • Amphibians & Reptiles – 10% Holtfreter’s solution [46] • Amphibians & Reptiles – Ca+2, Mg+2-free 10% Holtfreter’s solution [46] • Avians – Tyrode’s balanced salt solution, #T-2145 (Sigma) • Avian Ca+2, Mg+2-free – Tyrode’s balanced salt solution, #T-2145 (Sigma) • Non-human mammals – Phosphate Buffered Saline, PBS (Sigma) • Non-human mammals – Ca+2, Mg+2-free Phosphate Buffered Saline, PBS (Sigma) • Humans – Dulbecco’s Phosphate-Buffered Saline (10X) #310-4080AJ (GIBCO) • Humans – Dulbecco’s Phosphate-Buffered Saline (10X) #310-4080AJ (GIBCO) 2.1.2. Enzymes • Trypsin, Sigma • Collagenase type-1, Worthington Biochemicals • Dispase, R&D Laboratories 2.1.3. ELICA Fixative Reagents • Species-Specific Buffers • Paraformaldehyde, #P6146, Sigma • Glutaraldehyde, #G5882, Sigma • Sodium Azide, #S2002, Sigma • D-Glucose, # G-6138, Sigma 2.1.4. Inhibition and Exhaustion of Endogenous Peroxidases • 5% Sodium Azide, #S2002 Sigma • 30% Hydrogen Peroxide, #H-1009, Sigma 2.1.5. 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 GSC Advanced Research and Reviews, 2025, 25(02), 407-440 422 Figure 8 When plating aTPSCs always perform front to back and side to side movements of culture vessel to obtain an even coating of cells across plate surface. Due to centrifugal force, a circular motion will create micro-mass clumps of cells around the periphery of the flask which will alter the final results of the experiment 2.2.11. Propagation Cultures were fed whenever the color of the medium changed from salmon-colored to orange-yellow in color (Fig. 7) [65]. To maintain a standardized propagation time (doubling time + > 2 hours), 2-ng per ml platelet-derived growth factor-BB was added to the in-house medium [76]. 3. Procedures - methodologies 3.1. Animal Use The use of animals in this study complied with the guidelines of Mercer University’s IACUC. 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 [83]. 3.2. Tissue Harvest from Skeletal Muscle Utilizing sterile procedures with universal precautions, postnatal male and female Sprague-Dawley rats (200-300 g) were euthanized using carbon dioxide inhalation [81,82]. The rats were soaked with 70% ethanol and transferred to a Class-2 Biosafety Cabinet. The hind limbs were washed with a Betadine solution, a circular incision made around the juncture of the thigh to the trunk of the body, and the skin pulled down over the foot. The bellies of the anterior compartment leg muscles, e.g., tibialis anterior, extensor digitorum longus; lateral compartment muscles, e.g., fibularis longus and brevis; and posterior compartment muscles, e.g., soleus, gastrocnemius, tibialis posterior, and flexor digitorum longus, were separated from their tendon attachment sites [83]. The removed skeletal muscle bellies were pooled in species-specific buffer in sterile pre-tared 50-ml polypropylene centrifuge tube and weighed. The skeletal muscle in species-specific buffer was then aliquoted into glass petri dishes, no more than 5-g per dish, and minced to the consistency of orange marmalade using sterile watchmaker’s forceps with a pre-sterilized (wiping down outside surfaces with 70% ethanol) stereo-dissection microscope [65]. The resultant minced tissue was returned to sterile 50ml polypropylene centrifuge tubes and enzymatically digested with collagenase/dispase [78]. The enzymatic solution consisted of 250 units per ml of type-I collagenase, 33.3 units per ml of dispase, 1% antibiotic-antimycotic, in calcium+2free, magnesium+2-free Opti-MEM + GlutaMax, pH 7.4 [64]. The caps were tightened. Parafilm was used to seal the tubes. GSC Advanced Research and Reviews, 2025, 25(02), 407-440 423 The 50-ml tubes were placed in an Orbital Incubator Shaker water bath (Labline) at a speed of 100 rpm for 18 hours at 37oC. After 18 hours of digestion the tubes were removed from the incubator/shaker, the outside of the tubes disinfected with 0.5% v/v Amphyl solution (VWR, Bristol, CT) in deionized water. The 50-ml tubes were centrifuged at 25 RCF (Relative Centrifugation Force) for 10 minutes. The tubes were checked for pelleted material. In some instances, undigested tissue remained in the tubes. The supernatants were removed and placed into fresh 50-ml conical tubes and centrifuged at 1800 RCF for 10 minutes. The supernatants were discarded. The pelleted cells were re-suspended by agitation and trituration. The dispersed cell pellets were reconstituted with 2-ml of basal medium. The basal medium consisted of Opti-MEM + GlutaMax, 10% Heat Inactivated Serum (Atlas Biologicals), 1% antibiotic-antimycotic (Sigma), pH 7.4. Ten ng/ml PDGF-BB (R&D Systems) was added to the basal medium to form the propagation+ medium [13,23]. Cell counts were performed as described below. 3.3. Cell Counts The final volume of each cell suspension was measured and recorded. Fifteen microliters of each cell suspension were removed and placed into separate 2.0 ml polypropylene tubes (Corning). Fifteen microliters of sterile 0.4% Trypan Blue solution (filter-sterilized) 0.4 g Trypan blue (Kodak) in sterile PBS was added to each tube. The contents were mixed by trituration 5-6 times and 15 microliters of the cell suspension/Trypan blue solution was placed on a hemocytometer for cell counting. All ultra-small Trypan blue-positive cells, small Trypan blue-positive/negative cells, and Trypan blue negative cells (Fig. 1) within the nine large boxes of the hemocytometer were counted separately and then averaged for the number of cells of each type per each large box. The isolated cells were counted on a hemocytometer by four separate individuals, the counts averaged and standardized per gram of tissue. The final cell number calculations were based on number of cells per gram of tissue. The 0.4% Trypan blue that is commercially available is pre-made in water, which lyse a majority of the cells, due to water’s low osmotic pressure. For accurate cell counting it is far better to make sterile 0.4% Trypan blue in speciesspecific buffer, pH to 7.4, and store in an opaque/brown bottle at ambient temperature [65]. The species-specific buffer of choice for amphibians and reptiles is 10% Holtfreter’s solution [84,85]; for avians, it is Tyrode’s buffer, pH 7.4 [60-61]; for non-humans it is Phosphate Buffered Saline [62]; and for humans, it is Dulbecco’s Phosphate Buffered Saline, Ph 7.4 [63]. Sterilized 0.4% Trypan Blue is made by weighing out 0.4 grams of dry Trypan Blue powder, adding to 100-ml of species-specific buffer of choice, and sterilized by filtering through a 0.1-micron bottle top filter (Thermo-Fisher). Sterilized Trypan blue staining solution should only be used in a sterile hood, and never outside the hood on the countertop. Otherwise, the possibility of bacterial contamination is very high, which can lead erroneous results with respect to the number of viable cells obtained [65]. 3.4. Electrostatic Charge of Surface Plastic of Culture Vessels The reason for the differences with respect to culture vessels from different manufacturers is that the manufacturers used plastics with different electrostatic charges for their various culture vessels. The plastic culture vessels examined were composed of plastic with a surface electrostatic charge of either negative, neutral, or positive (Fig. 6) [65]. Initially, we attempted to obtain the plastic electrostatic charge information from technical representatives from the various companies. Most either had no clue what we were asking or would not tell us the information, citing proprietary information that could not be divulged. Therefore, this side study was obtained through brute force testing. After testing various sized culture vessels, from a plethora of companies, the particular culture vessels chosen for use with aTPSCs, circa 1988-2008, were 96-well plates (Costar), 48-well plates (Corning, Corning, NY), 24-well plates (Corning), 6-well plates (Corning), T-25 flasks (Falcon, Thermo-Fisher Scientific), and T-75 flasks (Falcon) [13,23,65]. Knowledge of whether the plastic composition for these plates has changed is currently unknown. But given the above troubleshooting notes, it is relatively easy to ascertain. 3.5. Substratum for Attachment The original procedure for making the various percent concentrations of type-1 collagen for testing followed established protocols, e.g., measure designated gram quantities of type-1 collagen peptides (Sigma-Aldrich Chemical), was to add dry material w/v to 100-ml sterile reverse osmosis double-distilled water, stir contents for 48 hours, then sterile-filter through a 0.1-micron filter. 96-well plates were coated with resultant percentages of type-1 collagen, and allowed to dry for 24-hours. Cells suspended in plating medium at 10^3 cells/well were plated into each well containing the percentage collagen coating solutions, e.g., 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5, and 10%, with n=12 per percent coating. Cells were allowed to attach for 24 hours, and medium removed. The DNA content of each well, noting number of cells attached, was measured using the ELICA procedure [23]. GSC Advanced Research and Reviews, 2025, 25(02), 407-440 424 For our studies, a less arduous and less time-consuming procedure for 1% collagen coating was developed. Add 1-g of type-1 collagen peptides to w/v to 100-ml HPLC water, but don’t stir. Autoclave the solution on the liquid cycle for 15 min. Remove from the autoclave and let solution in bottle return to ambient temperature (22-39oC, warm to the touch) on the countertop. The 1% collagen solution should be water crystal clear. If one tries to hasten the cooling process with refrigeration or an ice bath, the collagen peptides will precipitate out of solution (become cloudy) and the cells will not attach to the resultant substratum [65,76]. The culture vessels were coated with the 1% collagen solution at regular intervals at the same time and then stored in a closed cabinet at ambient temperature for future use. In a HEPA-filtered Class-2 Biosafety Cabinet, pipet a volume of 1% collagen solution that is ½ the maximum medium feeding volume for each vessel. For example, maximum feeding volume in each well of a 96-well plate is 200-microliters. Therefore, pipet 100-microliters of 1% collagen solution into each well. Place lids/caps on the vessels. Move vessels back and forth and side to side, the same as when plating cells, providing for an even distribution of the collagen solution across the vessel surface (Fig. 7). Allow it to sit undisturbed in the Biosafety Cabinet for a minimum of 30-min with UV-light turned on. Turn off UV light and aspirate residual 1% collagen solution into bleach using a sterile 9-inch glass pipet attached to a vacuum apparatus. Cover vessels with lids/caps and store in a cabinet at ambient temperature. Shelf life for 1% collagen coated culture vessels is approximately one year. However, due to the number of experiments that were performed, collagen coating usually occurred every month, depending on the vessels utilized. Usually, a case of each vessel was coated at a time. Multiple 100-ml batches of 1% collagen solution for coating flasks were generated, based on the number of vessels being coated and the volume of the solution to be used [63]. Any leftover sterile 1% collagen solution was saved at ambient temperature (never refrigerated), and used inside a sterile class-2 hood to negate collagenase/dispase enzymatic digestion of substrate to release the cells from tissues or culture vessels [13,23,65,76]. 3.6. Preferred Medium for aTPSCs Culture medium of choice for aTPSCs is OptiMEM + GlutaMax (GIBCO). When cells are cultured for in vitro testing using the ELICA procedures, antibiotic/antimycotic, e.g., penicillin, streptomycin, and fungizone, are added to maintain sterility in the cultures [65]. When cells were propagated for in vivo testing, antibiotic/antimycotic was withheld from the medium. 3.7. Anerobic Environment Beta-mercaptoethanol was added to culture medium of propagating mammalian cells (TSCs, HLSCs, CLSCs, and PSCs) destined for characterization to reduce oxygen tension, e.g., 5% O2, 5% CO2, 90% N2, then pH to 7.4. In contrast, GLSCs, EctoSCs, MesoSCs, EndoSCs, and MSCs destined for characterization with biological agents (inhibition, proliferation, progression, and induction) preferred an ambient oxygenated environment (21% O2, 5% CO2, 74% N2), then pH to 7.4 [13,23,65]. If human cells are cultured for use in clinical studies/trials, the medium must be absent of any antibiotic/antimycotic, fungizone, beta-mercaptoethanol, and/or putrescine. Therefore, a sterile containment unit, BioSpherix, was utilized [68]. This unit contains a HEPA-filtered Class-2 hood for ingress and egress of supplies, multiple sterile chambers for culturing, cell processing, centrifugation, and a computer system that monitors and regulates CO2, O2, N2, floor temperature, chamber temperatures, particle counts, volatiles, etc., in the various chambers. 3.8. Serum Depending on the particular lot of serum that was used, there were differences within the cells with respect to resultant phenotypic expression markers. Most of the sera contained biological agents that stimulated the formation of fibroblasts in the TSC and PSC cultures [67]. And these particular serum (or recombinant protein) fibroblastic agents induced the TSC, PSC, and MesoSC cultures to form an ECM reminiscent of scar fibroblasts/fibrocytes [31,65]. When recombinant proteins were tested individually, it was noted that both TGF-beta and basic-FGF would induce similar scar fibroblastic phenotypes and scar-ECMs. TGF-beta and basic-FGF are two of the most prominent components in platelets, as assessed by SDS-PAGE of 46 bands of proteins from lysed platelets seen with Coomassie blue staining, transferred to nylon membranes, and probed with antibodies to inductive agents (data not shown) [66]. It is conceivable that platelets were lysed during the commercial collection of serum releasing these components into the serum. To do any type of repetitive testing for biological activity, one needs to maintain the cells in their own undifferentiated state. Initially, when single cell repetitive serial dilution cloning was performed in chicken cells, over 400 lots of serum from bovine, calf, horse, pig, and human were screened. Only a single lot of serum (HS7, horse serum, 7th lot tested) was discovered to be void of any of the four biological agents affecting the cells, e.g., inhibition, proliferation, progression, or induction [13]. The entire lot was purchased and stored at -80oC for testing. Horse serum HS7 was used for cloning avian cells and initial testing of human recombinant proteins for biological activity [13,23,38,65,67,69,70]. For cloning mouse GSC Advanced Research and Reviews, 2025, 25(02), 407-440 425 stem cells, a heat inactivated serum procedure was devised to negate the potential biological activities of factors inhibiting differentiation, proliferation, progression, and induction [69]. The process for heat inactivation was to incubate any serum from bovine, fetal calf, equine, porcine or human origin, at 56oC for 8 hours, high speed centrifugation at 200,000 RCF to remove precipitate, and then positive pressure filtration through a 0.1-micron filter to sterilize [13,23,65]. For cloning rat cells and culturing human cells for study, the heat inactivation technique was given to Atlas Biologicals, in Fort Collins, CO to generate the heat inactivated serum [13], which was/is commercially available. Ten percent Heat Inactivated serum (Atlas Biologicals) was added to the cultures to maintain growth of the cells [67]. 3.9. Doubling Rate and Potential for Mutations The doubling rate for telomerase positive TSCs require 12-14 hours; HLSCs and CLSCs require 13-15 hours; PSCs require 14-16 hours; GLSCs require 16-18 hours; EctoSCs, MesoSCs, and EndoSCs require 18-24 hours; while telomerase negative MSCs require days to double in number (Table 1) [13]. We titrated platelet-derived growth factorBB (PDGF-BB, R&D Systems) to its physiological concentration (2-ng/ml) in basal medium and designated it as propagation+ medium to maintain established proliferation rates (doubling rate + > 2-hours) for propagation of the cells without mutations [65,76]. 3.10. Cell Release from Tissue Culture Vessel The aTPSC release from culture vessels occurs sequentially in two phases. The first phase entailed incubation with EGTA (ethylene-glycol-tetraacetic acid, a specific calcium chelator, Sigma) dissolved in an electrolyte-free (calcium, magnesium, potassium, and sodium) buffer of choice [86]. This disrupted the calcium-dependent attachment to its surrounding ECM, allowing the cells to round up with minimal attachment sites to the substratum. The second phase consisted of 250-units of type-1 collagenase and 33.3-units of dispase suspended in electrolyte-free EGTA buffer of choice. The protocol for release from the surrounding ECM is to remove culture medium from the culture vessel; rinse cultures twice with species-specific buffer to remove any residual material; aspirate wash solution into bleach; add electrolyte-free EGTA-buffer to vessel at ½ volume of feeding medium. Look through a phase contrast microscope as cells round up as they are released from their calcium dependent binding sites. Provide a mild rocking motion if required. This EGTA incubation process takes from 1-5 minutes and is dependent on the cell types released. The smaller the cell type, the lesser the number of calcium-dependent binding sites, the shorter the time frame for release from calcium-dependent sites, the quicker the rounding up of the cells. The electrolyte-free EGTA-buffer solution is removed and replaced with electrolyte-free EGTA buffer containing type-1 collagenase and dispase. This solution usually takes approximately 30-60 seconds for cells to detach completely from the culture vessel surface either as single cells or as sheets of cells. Triturate the solution to suspend released cells in enzyme solution and remove cell suspension from culture vessel into a 50-ml polypropylene centrifuge tube. The 1% type-1 collagen solution remaining from coating tissue culture vessels dilutes the cell suspension 14:1 and used to neutralize any further activities of the enzymes. To remove enzymes from cells, centrifuge the cell suspension to form a cell pellet. Decant supernatant into 10% sodium hypochlorite solution (bleach) and then using repeated trituration, resuspend cells in complete culture plating medium of choice. Preferential culture plating medium for TSCs and PSCs in mammalian species is Opti-MEM plus GlutaMAX (stabilized form of glutamine, GIBCO), 10% Heat Inactivated Serum, 1% antibiotic/antimycotic, 0.009% betamercaptoethanol, at pH 7.4. Preferential culture plating medium for GLSCs, EctoSCs, MesoSCs, and EndoSCs in mammalian species is Opti-MEM plus GlutaMAX (stabilized form of glutamine, GIBCO), 10% Heat Inactivated Serum, 1% antibiotic/antimycotic, at pH 7.4. 3.11. Plating Cells The mixed populations of cells, e.g., aTPSCs, progenitor cells, and differentiated cells, were plated onto various sized 1% collagen-coated tissue culture vessels [13,23], based on the number of cells per ml of basal medium. Plating density for each well of 96-well plates was 10^3 cells per 0.1-ml. Plating density for T-25 and T-75 flasks was 2x10^5 cells per ml, with 5-ml plated into T-25 flasks and 10-ml plated into T-75 flasks. When plating cells, swirling the culture vessels after cell/medium deposition will cause the cells to move to the periphery by centrifugal force, forming uneven plating of cells across vessel surface (Fig. 8). The swirling action predominantly forms micro-mass cultures of cells, which will lead to erroneous differentiation results [87-90]. Therefore, once cell suspension/medium was added to culture vessels the vessels were rocked side to side and back to front (Fig. 8). This allows for an even distribution of the cells across the vessel surface [13,23]. Twenty-four hours after plating, the plating medium was switched to propagation medium. Propagation medium is plating medium with the addition of 2-ng/ml PDGF-BB. Thereafter, propagation medium changes were based on the color change of the medium from salmon-colored to orange-yellow (Fig. 7). The time between medium changes ranged GSC Advanced Research and Reviews, 2025, 25(02), 407-440 426 from days to hours, dependent on the number of cells being propagated. When cultures reached confluence (telomerase negative differentiated cells, telomerase negative progenitor cells and/or telomerase positive germ layer lineage stem cells) or multi-layered post-confluence (telomerase positive pluripotent stem cells and telomerase positive totipotent stem cells), the cells were released and replated onto fresh 1% collagen-coated dishes. 3.12. Propagation of Cells Regardless of cell type, e.g., telomerase negative differentiated cells, telomerase negative progenitor cells, or telomerase positive germ layer lineage stem cells, telomerase positive pluripotent stem cells, or telomerase positive totipotent stem cells, as long as the cultures were fed with fresh medium when medium color changed from salmon-color to orangeyellow, the cells would survive (Fig. 7) [65]. To maximize cell numbers and dependent on the cells being propagated, cells were released and replated into fresh 1% collagen-coated dishes in plating medium, then switched to propagation+ medium 24-hours after plating. For differentiated cells, progenitor cells and germ layer lineage stem cells, when cultures reached single-layer confluence, they were released and replated (Fig. 9). For pluripotent stem cells (Fig. 10) and totipotent cells (Fig. 11), cultures were allowed to propagate past well confluence (600-800%) before release and replating. 3.13. Plating Plating followed by replating continued until sufficient numbers of cells were obtained for segregation into individual populations of differentiated cells, progenitor cells, and aTPSCs, by differential cryopreservation and cell sorting. 3.14. Release from Culture Vessel First, the medium was decanted into bleach and the vessel was rinsed with a magnesium-free and calcium-free speciesspecific buffer to remove any unattached constituents. Next, initially we used ethylenediamine-tetraacetic acid (EDTA), but later switched to ethylene glycol-tetraacetic acid (EGTA) and dissolved in magnesium-free, calcium-free speciesspecific buffer. While EDTA will chelate calcium, along with any other divalent cation, e.g., magnesium (Mg+2), manganese (Mn+2), zinc (Zn+2), copper (Cu+2), iron (Fe+2), or lead (Pb+2), EGTA is specific solely for calcium. The calcium chelator in species-specific calcium-free buffer [86] was used to disrupt the calcium-dependent binding site. Flattened cells would round up with only a few attachments to the substratum. Incubation took anywhere from 1-5 minutes depending on number of cells in the culture vessel, and directly proportional to the amount of disrupting buffer utilized. This was followed with incubation in an enzymatic solution containing type-1 collagenase and dispase to disrupt the ECM and basement membrane attachment sites to free the cells from the plate surface [13,23,65]. 4. Results Table 3 Species, Age and Location of Adult Telomerase Positive Stem Cells Ch1 Sa2 Rep3 Av4 Mo5 Rt6 Rb7 Fe8 Cn9 Ov10 Cp11 Pr12 Bo13 SB14 Eq15 HM16 HF17 Pre18 +19 + + Mor 20 SEM 21 Psn22 + + + + + + + + + + + + + Nb23 I24 I + + Ad25 I I + + SexM26 H27, Hc28 I I + + Ge29 I30 I31 I32 I33 SkM34 H, Hc H, Hc, I H, Hc, I, Cr35 H, Hc, I, Cr H, Hc I I I I I H, Hc, I H, Hc, I H, Hc, I GSC Advanced Research and Reviews, 2025, 25(02), 407-440 427 Der36 H, Hc I I, Cr I, Cr I, Cr I I Hrt37 H, Hc I I I, Cr SmM38 H, Hc I I + + Gt39 I I I Bone40 H, HC H, HC H, HC H, HC H, HC Pos41 Hh I I I, Cr Cr I I HyCart42 H, HC H, HC H, HC H, HC H, HC ElasCar43 H, HC H, HC H, HC H, HC ArtCart44 H, HC H, HC H, HC H, HC H, HC FibCart45 H, HC H, HC H, HC H, HC GPCart46 H, HC H, HC Pch47 H, HC I I I, Cr Cr I I Ns48 H, Hc I I I, Cr Cr I I Adip49 H, Hc I I I, Cr Cr I I Lig50 H, Hc I I I, Cr Cr I I Ten51 H, Hc I I I, Cr Cr I I BV52 H, Hc I I I, Cr Cr I I BoM53 H, Hc I I I, Cr Cr I I Bld54 Pre-GC55 I I Bld Post-GC56 I I I I I I I I I I I I I I I Tra57 I, Cr Cr Lng58 I, Cr Cr Eso59 I, Cr Cr Stm60 I, Cr Cr Liv61 I, Cr Cr SmI62 I, Cr Cr LgI63 I, Cr Cr SpI64 I, Cr Cr Brn65 I, Cr Cr Men66 I, Cr Cr SpC67 Cr Pan68 I, Cr Cr I I Kid69 I, Cr Cr GSC Advanced Research and Reviews, 2025, 25(02), 407-440 428 Ub70 I, Cr Cr Thy71 I, Cr Cr Tng72 I, Cr Cr Cr Cr Tes73 I, Vi74 Ov75 Cr Ft76 Cr Kar77 Dip78 Dip Dip Dip Dip Dip Table 3. Ch1, Characteristics; Sa2, adult terrestrial salamanders, Ambystoma maculatum, Ambystoma annulatum, Ambystoma texanum, Ambystoma tigranum; Rep3, reptile, Komodo Dragon; Av4, avians, Gallus domesticatus, Wedel Crane; Mo5, mouse; Rt6, rat: outbred Sprague-Dawley and inbred Wistar Furth; Rb7, rabbit; Fe8, feline (cat); Cn9, canine (dog); Ov10, ovine (sheep); Cp11, caprine (goat); Pr12, porcine (pig); Bo13, bovine (cow); SB14, Spectacled Bear; Eq15, equine (horse); HM16, human male; HF17, human female; Pre18, pre-natal; +19, presence of TSCs and PSCs; Mor20 , morula; SEM21, scanning electron microscopy of Day 8+ blastomeres; Psn22, Post-natal (after birth); Nb23, Newborn; I24, Isolation; Ad25, Adolescent; SexM26, sexually mature; H27, histology; Hc28, histochemistry; Ge32, geriatric; I30, isolation from NIH’s aging model, 36-month old CBF-1 mouse; I31, isolated from a 40 year old horse; I32, isolated from a 80 year old male with Parkinson’s disease; I33, isolated from a 98 year old female with Alzheimer’s disease; SkM34, Skeletal Muscle; Cr35, cryosection followed by immunocytochemistry using antibodies to IA4 (smooth muscle, positive control), CEA-CAM-1 (TSC) and SSEA-4 (PSC); Der36, dermis of skin ; Hrt37, heart; SmM38, smooth muscle; Gt39, granulation tissue; Bone40, bone; Pos41, periosteum; HyCart42, hyaline cartilage; ElasCart43, elastic cartilage; ArtCart44, articular cartilage; FibCart45, fibrocartilage; GPCart46, growth plate cartilage; Pch47, perichondrium; Ns48, nerve sheaths; Adip49, adipose tissue (unilocular white fat); Lig50, ligament; Ten51, tendon; BV52, arterial and venous blood vessels; BoM53, bone marrow (hematopoietic cells and stromal cells); Bld54, blood pre-GC55, before glacial cap ingestion if individual had pre-existing morbidity(ies); Bld post-GC56, Blood after glacial cap ingestion; Tra57, trachea; Lng58, lung; Eso59, esophagus (lamina propria, submucosa, adventitia); Stm60, stomach (submucosa, serosa); Liv61, liver; SmI62, small intestine (lamina propria, submucosa, serosa); LgI63, large intestine (lamina propria, submucosa, serosa); Spl64, spleen (capsule, trabeculae, interstitial tissue); Brn65, brain (white mater, gray mater); Men66, meninges (dura mater, arachnoid mater, pia mater); SpC67, spinal cord (white mater, gray mater); Pan68, pancreas (exocrine and endocrine portions); Kid69, kidney (capsule, interstitium); Ub70, urinary bladder; Thy71, thyroid; Tng72, tongue; Tes73, testis; Vi74, vibratome sectioning followed by immunocytochemistry; Ov75, Ovary; FT76, fallopian tube; Kar77, karyotype; Dip78, respective diploid number of chromosomes for each species examined [31]. Four additional species were added to the Table: Komodo Dragon, Wedel Crane, Spectacled Bear, and German Shepard, see reference [45]. Reprinted with permission from Young HE, Black AC. Pluripotent Stem Cells, Endogenous versus Reprogrammed, a Review. MOJ Orthop Rheumatol 1(4): 00019, 2014 [31]; Young HE, Speight MO. Osteoarthritis Treated with Telomerase-Positive Adult Stem Cells in Animals and Humans. Stem Cells Regen Med. 2020; 4(2):1-11 [45]. 4.1. Plating a Mixed Cell Isolate The aTPSCs, specifically PSCs and TSCs, and a progenitor cell are seen in a plated mixed cell population derived from fresh isolates using type-1 collagenase/dispase enzymatic digestion of solid tissues, 24-hours after plating (Fig. 5). With time, the progenitor cells reach confluence and are overlain with PSCs and TSCs (Fig. 9). With continued time in culture, and due to the doubling rate of PSCs and TSCs, the progenitor cell cultures can become overrun with PSCs and TSCs (Fig. 10) to a point that even the PSCs can become overrun with TSCs (Fig. 11). GSC Advanced Research and Reviews, 2025, 25(02), 407-440 429 Figure 9 Single contact inhibited layer of progenitor cells (asterisk) overlain with pluripotent stem cells (PSCs, large arrowheads), halo-like stem cells (HLSCs, arrows), and TSCs (3 small arrowheads). Visible multiple cell types (contact inhibited progenitor cells, TSCs, HLSCs, and PSCs) suggest contaminating cells within a “pure” culture of contact inhibited progenitor cells [65]. Reprinted with permission from Young HE. Endogenous Adult Telomerase Positive Stem Cells and/or Combinatorial Nutraceuticals, 50 Years in the Making, 50 Years of Discovery; (In preparation) Figure 10 Initial plating of aTPSCs to form multi-layered confluent cells. Post-natal totipotent stem cells (TSCs) were initially plated onto a type-1 collagen substratum and allowed to grow to multi-layered confluence. Next, post-natal pluripotent stem cells (PSCs) were seeded onto layers of TSCs to form a bilaminar shell of cells [39]. Reprinted were permission from Young HE, Limnios JI, Lochner F, McCommon G, Cope LA, Black AC Jr. Pancreatic islet composites secrete insulin in response to a glucose challenge. J Stem Cell Res. 2017; 1(1) 001: 1-12 GSC Advanced Research and Reviews, 2025, 25(02), 407-440 430 Figure 11 post-natal pluripotent stem cells (PSCs) plated on decellularized pancreatic collagen substratum (upper portion of photograph). Outer layer of 3D organoid consisted of post-natal totipotent stem cells (TSCs) (lower portion of photograph) plated on top of PSCs, eventually forming a bilaminar layer of cells [39]. Reprinted were permission from Young HE, Limnios JI, Lochner F, McCommon G, Cope LA, Black AC Jr. Pancreatic islet composites secrete insulin in response to a glucose challenge. J Stem Cell Res. 2017; 1(1) 001: 1-12 5. Discussion The maintenance progenitor cells were originally designated as “adult stem cells”, because they were from post-natal individuals and would form one or more cell types with a separate identity from themselves [13,91]. This is a misnomer in terminology for several reasons. 1. There is loss of the telomerase enzyme in maintenance progenitor cells at birth. Therefore, these cells have a finite lifespan in humans of 50-70 population doublings [92] and in rodents of 6-8 population doublings [93] before cell senescence and cell death [94]. 2. Maintenance progenitor cell numbers decrease with increasing age of the animal, including humans [19]. 3. Maintenance progenitor cells are pre-committed to distinct differentiated cell types [13,23]. 4. Maintenance progenitor cells are responsive to progression factors that accelerate their expression to their pre-determined cell types [65]. And 5. Maintenance progenitor cells are unresponsive to known inductive factors (human recombinant proteins) outside their pre-determined cell types [65]. Examples of the healing stem cells are the endogenous adult telomerase positive stem cells (aTPSCs) [13]. The eight populations of aTPSCs are uniquely different from each other as well as being uniquely different from both functional differentiative cells and maintenance progenitor cells as shown in Tables 1 and 2. These differences include, but not limited to, size, 0.4% Trypan blue staining patterns, cell surface markers, age range (Figs. 1-3), growth in culture (Figs. 10-13), cryopreservation temperatures, population doublings, and differentiation capabilities (Tables 1 and 2) [13]. Healing stem cells are telomerase positive which gives them an essentially unlimited proliferation potential [94]. There are eight distinct subcategories of healing stem cells based on multiple parameters, including differentiation potentials (Figs. 2,3) [13]. In contrast to maintenance progenitor cells: 1. Healing stem cells (aTPSCs) retain the telomerase enzyme at birth, giving the cells essentially an unlimited lifespan as long as they remain undifferentiated [95]. 2. Healing stem GSC Advanced Research and Reviews, 2025, 25(02), 407-440 431 cells remain constant throughout the lifespan of the organism, from four-cell stage embryogenesis to geriatric-aged individuals, as long as the cells remain undifferentiated (Fig. 4, Table 2) [13,31]. 3. Two categories of healing stem cells exist with different pre-commitments. The aTPSCs that will form all somatic cells of the body are composed of TSCs, HLSCs, CLSCs, PSCs, and GLSCs. Healing stem cells that are pre-committed to particular germ layer lineages are the EctoSCs, MesoSCs, and EndoSCs [13,23,65]. 4. Healing stem cells are not responsive to progression factors [65]. And 5. Healing stem cells are responsive to both known and “unknown” inductive factors, e.g., human recombinant proteins, morphogenetic proteins, and exosome-conditioned medium derived from differentiated cells [65]. In situ, healing stem cells reside in connective tissue niches interspersed throughout the body as dormant, hibernating, quiescent cells [65]. They can be stimulated to proliferate and mobilize into the blood stream as quiescent non-activated cells [96], but they need to be activated to function for replacing damaged cells and tissues [97]. Activation occurs by unmasking homing receptors on their cell surface and unmasking cell surface receptors for biological agents that direct the ultimate fate of the cells. Within an individual, activation of homing receptors occurs with release of chemokines from damaged tissues [98,99], and activation of receptors for exosomes occurs with endogenous matrix metalloproteinases at the site of injury [100]. Due to their limited number in the body and their uniqueness in not conforming to established tissue culture practices [34], special methodologies were developed to examine them both in vitro and for their use in vivo. This begins a series of articles explaining in step-by-step detail the methodologies (e.g., rationale, standardization, reagents, manufacturers, catalog numbers, step-by-step instructions, expected results) that were developed for in vitro characterization and in vivo use. The current report describes the methodologies developed to optimize the viability of the aTPSCs during their isolation, plating, and propagation for in vitro analyses. 5.1. Location in the Body As shown (Fig. 4), three pairs of small (0.1-2.0 micron) and one comparatively larger (6-8 micron) cellular structures appear interspersed within 8+-cell stage blastomeres of a developing embryo [31]. Their appearance early in development explains their location within all connective tissues of a post-natal individual, within the umbilical cord (data not shown), and within the placenta (data not shown). The latter two structures, e.g., umbilical cord and placenta, form after the 8-cell stage during embryogenesis [101]. In post-natal individuals, biopsy specimens from newborn to geriatric individuals were obtained, immersed in ELICA fixative for preservation, sectioned, and stained using the ELICA procedure for immunochemistry of tissue sections using antibodies for TSCs (CEA-CAM-1), PSCs (SSEA-4) [102]. Procedural controls were added to verify that the staining for TSCs and PSCs was real and not an artifact of preparation. Each ELICA staining run contained positive and negative controls to validate that the procedures were working properly. An antibody to smooth muscle alpha-actin (IA4) was used as the positive control [23,102]. Six negative controls, one for each step in the ELICA procedure [23,102], were utilized to demonstrate the potential for non-specific binding of the reagents to the tissue or to each other. Immuno-stained representative figures (Figs. 3-17 in [24]) show SSEA-4+ cells (PSCs) and CEA-CAM-1+ cells (TSCs) located within the connective tissue compartments of their associated organs, e.g., heart (pericardium, coronary vessels, intramural septum), brain, pancreas, dermis, adipose tissue, lung, spleen, bone marrow, kidney, skeletal muscle, testis, ovary, and fallopian tube [24]. In toto, biopsy specimens from newborn to geriatric-aged individuals, comprising multiple organs from 15 species of animals, including humans, were assayed for location of TSCs and PSCs within an individual. The results demonstrate that TSCs and PSCs are a conserved population of cells present within the connective tissues of all animals examined (Table 2) [31,45]. 5.2. Harvesting Cells from Solid Tissues The standard procedure for harvesting cells from solid tissues is to macerate the tissue and then treat with trypsin for 15 min at 37oC to release the cells [34]. Trypsin cleaves proteins between adjacent lysine and arginine residues. Therefore, it will disrupt the core proteins of proteoglycans and glycoproteins within the extracellular matrix, thus releasing the cells. Unfortunately, cell membranes also contain adjacent lysine and arginine residues, and are disrupted causing loss of cell integrity and decreased viability of released cells [58-61,65]. An alternate method was developed to optimize viability of the aTPSCs during the harvesting procedure. This entailed using type-1 collagenase and dispase in combination to disrupt just the extracellular matrix constituents releasing the cells from their connective tissue niches [13,65]. 5.3. Counting Cells Cell counting occurred using sterile 0.4% Trypan blue in species-specific buffer [23]. The commercially available 0.4% Trypan blue is pre-made in water, which will lyse a majority of the cells, due to water’s low osmotic pressure [65]. It is GSC Advanced Research and Reviews, 2025, 25(02), 407-440 438 [60] Young HE, Ceballos EM, Smith JC, Lucas PA, Morrison DC. Isolation of embryonic chick myosatellite and pluripotent stem cells. Journal of Tissue Culture Methods, 14:85-92, 1992. [61] Young HE, Ceballos EM, Smith JC, Mancini ML, Wright RP, Ragan BL, Bushell I, Lucas PA. Pluripotent mesenchymal stem cells reside within avian connective tissue matrices. In Vitro Cellular & Developmental Biology. 1993; 29A:723-736. [62] Pate DW, Southerland SS, Grande DA, Young HE, Lucas PA. Isolation and differentiation of mesenchymal stem cells from rabbit muscle. Surgical Forum. 1993; XLIV:587-589. [63] Young HE, Steele T, Bray RA, Hudson J, Floyd JA, Hawkins K, Thomas K, Austin T, Edwards C, Cuzzourt J, Duenzl M, Lucas PA, Black AC Jr. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec. 2001; 264:51-62. [64] Stout CL, Ashley DW, Morgan III JH, Long GF; Collins JA, Limnios JI, Lochner F, McCommon G, Hixson D, Black Jr AC, Young HE. Primitive stem cells reside in adult swine skeletal muscle and are mobilized into the peripheral blood following trauma. American Surgeon. 2007; 73 (11):1106-1110. [65] Young HE. Endogenous Adult Telomerase Positive Stem Cells and/or Combinatorial Nutraceuticals, 50 Years in the Making, 50 Years of Discovery; In preparation [66] Lucke’ B, McCutcheon M. The living cells as an osmotic system and its permeability to water. Physiol Rev. 1932; 12(1): 68-139. [67] Young HE, Duplaa C, Romero-Ramos M, Chesselet M-F, Vourc’h P, Yost MJ, Ericson K, Terracio L, Asahara T, Masuda H, Tamura-Ninomiya S, Detmer K, Bray RA, Steele TA, Hixson D, El-Kalay M, Tobin BW, Russ RD, Horst MN, Floyd JA, Henson NL, Hawkins KC, Groom J, Parikh A, Blake L, Bland LJ, Thompson AJ, Kirincich A, Moreau C, Hudson J, Bowyer III FP, Lin TJ, Black Jr AC. Adult reserve stem cells and their potential for tissue engineering. Cell Biochem Biophys. 2004; 40(1):1-80. [68] www.BioSpherix.com [69] Rogers JJ, Adkison LR, Black AC Jr, Lucas PA, Young HE. Differentiation factors induce expression of muscle, fat, cartilage, and bone in a clone of mouse pluripotent mesenchymal stem cells. The American Surgeon. 1995; 61(3):231-236. [70] Young HE, Wright RP, Mancini ML, Lucas PA, Reagan CR, Black AC Jr. Bioactive factors affect proliferation and phenotypic expression in pluripotent and progenitor mesenchymal stem cells. 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[83] Animal Cell Culture Guide, The Essentials of Life Sciences Research, American Type Culture Collection, Gaithersburg, Maryland. [84] Humason G. Animal Tissue Techniques. WH Freeman and Co., San Francisco, CA. [85] Young HE, Dalley BK, Markwald RR. Effect of selected denervations on glycoconjugate composition and tissue morphology during the initiation phase of limb regeneration in adult Ambystoma. Anatomical Record. 1989; 223:223-230. [86] www.SigmaAldrich.com EGTA specific calcium chelator in reduced electrolyte buffer. [87] Paulsen DF, Solursh M. Microtiter micromass cultures of limb-bud mesenchymal cells. In Vitro Cell & Develop Biol. 1988; 24(2):138-147. [88] Schramm CA, Reiter RS, Solursh M. Role of short-range interactions in the formation of cartillage and muscle masses in transfilter-micromass cultures. Develop Biol. 1994; 163(2): 467-479. [89] Solursh M, Ahrens PB, Reiter RS. A tissue culture analysis of steps in limb chondrogenesis. In Vitro. 1978; 14(1): 51-61. [90] Swalla BJ, Solursh M. The independence of myogenesis and chondrogenesis in micromass cultures of chick wing buds. Develop Biol. 1986; 116(1): 31-38. [91] NIH Stem cell information page. In: Stem cell Information, Bethesda, MD, NIH, US DHHS, 2016. Cited July 28, 2020. [92] Hayflick L, Moorehead PS. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961; 25:585-621. [93] Rohme D. Evidence for a relationship between longevity of mammalian species and life spans of normal fibroblasts in vitro and erythrocytes in vivo. Proc Natl Acad Sci USA 1981; 78: 5009-5013. [94] Zvereva MI, Shcherbakova DM, Dontsova OA. Telomerase: structure, function, and activity regulation. Biochemistry (Mosc.). 2010; 75:1563-1583. [95] Young HE, Lochner F. Telomerase positive totipotent stem cells in the adult brain. I. cerebral cortex. Regen Med Biol Res 2021; 2(1):1-16. [96] Young HE. Fresh Isolate Adult Telomerase Positive Stem Cells: An addition to Embryonic Stem Cells (ESCs), Induced Pluripotent Stem Cells (iPSCs), and/or Mesenchymal Stem Cells (MSCs) for Regenerative Medicine. GSC Advanced Research and Reviews. 2023; 16(1):066-081. [97] Young HE. Combinatorial nutraceutical supplement pill (CNSP) stimulates naïve adult telomerase positive stem cells In Situ to reverse signs and symptoms in multiple human health concerns. GSC Advanced Research and Reviews. 2004; 20(02): 047-056. https://doi.org/10.30574/gscarr.2024.20.2.0290 [98] Bennett LD, Fox JM, Signoret N. Mechanisms regulating chemokine receptor activity. Immunology. 2011; 134 (3): 246-256. [99] Hughes CE, Nibbs RJB. A guide to chemokines and their receptors. FEBS J. 2018: 285(16): 2944-2971. [100] Costanzo RM, Perrino LA. Peak in matrix metalloproteinases-2 levels observed during recovery from olfactory nerve injury. Neuroreport. 2008; 19(3): 327-331. [101] Moore KL, Persaud TVN, Torchia MG. The Developing Human, Clinically Oriented Embryology, 9th Edition, 2013. ELSEVIER, Saunders. [102] Young HE. Carcinoembryonic antigen-cell adhesion molecule-1 and stage-specific embryonic antigen-4 are present in the reproductive organs of adult mammals. GSC Advanced Research and Reviews. 2025; 23(03): 149147. [103] Henson NL, Heaton ML, Holland BH, Hawkins KC, Rawlings B, Eanes E, Bozof R, Powell S, Grau R, Fortney J, Peebles B, Kumar D, Yoon JI, Godby K, Collins JA, Sood R, Bowyer 3rd FP, Black Jr AC, Young HE. Karyotypic analysis of adult pluripotent stem cells. Histology and Histopathology, 20: 769-784, 2005. GSC Advanced Research and Reviews, 2025, 25(02), 407-440 440 [104] Kierszenbaum AL. Histology and Cell Biology, An Introduction to Pathology. Elsevier/Mosby, 11830 Westline Industrial Dr, St Louis, MO 63146, Copyright 2002, ISBN: 0-323-01639-1 [105] Young HE, Black Jr AC. Adult stem cells. Anat. Rec. 276A:75-102, 2004. [106] Young HE, Black Jr AC. Naturally occurring adult pluripotent stem cells. In: Stem Cells: From Biology to Therapy, Advances in Molecular Biology and Medicine. 1st Ed, R.A. Meyers, Ed, WILEY-BLACKWELL-VCH Verlag GmbH & Co. KGaA. Chap 3, pp. 63-93, 2013. [Labeling differences between original paper and above, original paper Figure 4. ELSCs = PSCs and BLSCs = TSCs]. [107] Kumar V, Abbas AK, Fausto M, et al. In: Robbins and Cotran Pathologic Basis of Disease. Elsevier, Saunders, pgs. 226-230, 2010. [108] 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. [109] Human Use The use of human biopsy specimens in this study complied with the guidelines of Mercer University’s Institutional Review Board (IRB). These guidelines reflect the Federal Regulations for Protection of Human Research Subjects, HHS Office for Human Research Protections – 45 and 46 CFR: 46.102(I).