Adult Telomerase Positive Stem Cells: Remain Constant Throughout Life-Span of Individual

   

Henry E. Young^*1-6

Citation: Young HE. Adult Telomerase Positive Stem Cells: Remain Constant Throughout Life-Span of Individual. J Stem Cell Res. 7(1):1-13.

Received: January 21, 2026 | Published: January 30, 2026

Copyright© 2026 Genesis Pub by Young HE. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are properly credited.

DOI: https://doi.org/10.52793/JSCR.2026.7(1)-83

Abstract

Previous reports stated (implied) that (all) adult stem cells decrease within increasing age of an individual. Those reports utilized telomerase negative progenitor cells, such as mesenchymal stem cells (MSCs), and other progenitor cells to validate their results. Their manner of testing was to isolate their respective cells from different aged individuals using short term trypsin digests and then counting the released cells. Their results demonstrated a constant number of cells till about age thirty (in humans) and then a slow decrease in number of released cells with increasing age of the individual. The previously reported methods for assessing numbers of telomerase negative progenitor cells were recapitulated in a series of studies with adult telomerase positive cells using biopsy specimens from newborn, adolescent, post-pubescent, sexually-mature, and geriatric-aged individuals, utilizing short term trypsin digests, short term collagenase/dispase digests, and extended term collagenase/dispase digests keyed to the age of the individual. After the subsequent digests the released cells were counted. Both telomerase negative progenitor cells and telomerase positive cells could be released from the connective tissues of newborn, adolescent, post-pubescent, sexually-mature, and geriatric-aged individuals. The results suggest that while progenitor cells decrease with increasing age of the individual, telomerase positive cells remain constant throughout the life-span of an individual.

I would propose that the decrease in cell numbers of progenitor cells with increasing age of the individual relates to either the absence of the telomerase enzyme, thus limiting their population doublings with increasing age, or an age-based increase in the density of the extracellular matrix surrounding the cells.

Keywords

Stem Cells; Progenitor Cells; Decrease with Age; Constant with Age. 

Abbreviations

• 37⁰C, thirty-seven degrees centigrade
• aTPSCs, adult telomerase positive stem cells
• CEA-CAM-1, carcinoembryonic antigen-cell adhesion molecule-1
• CD, collagenase-dispase
• CLSC, corona-like stem cell
• DPBS, Dulbecco’s Phosphate Buffered Saline
• EctoSC, ectodermal stem cell
• EGTA, ethylene glycol bis (b-aminoethyl ether)-N, N, N’, N’- tetraacetic acid, a monospecific calcium chelator
• EndoSC, endodermal stem cell
• GLSC, germ layer lineage stem cell
• Ger, geriatric (85-years-old)
• HLSC, halo-like stem cell
• IRB, Institutional Review Board
• MAPCs, multipotent adult progenitor cells
• MesoSC, mesodermal stem cell
• MIAMIs, marrow-isolated adult multilineage inducible cells
• mm, millimeter
• mM, millimolar
• MSC, mesenchymal stem cell
• MUSEs, multilineage-differentiating stress-enduring cells
• NB, newborn, < 1-year-old
• NIH, National Institutes of Health
• NIRS, National Institute Research Services
• PSC, pluripotent stem cell
• Pub, pubescent (10-12-years of age)
• RCF, relative centrifugation force
• RGD, tripeptide Arginine (R), glycine (G), aspartic acid (D) sequence found in fibronectin, acting as a key recognition site for cell attachment, migration, and tissue development
• SM, sexually mature (30-years of age)
• SMS, small mobile stem cells
• VSELs, very-small embryonic-like stem cells
• T, trypsin
• TSC, totipotent stem cell

 

Introduction

The post-natal (adult) human is composed of trillions and trillions of cells. These cells can be divided into two categories: differentiated cells and precursor cells [1,2]. The differentiated cells are composed of functional parenchyma and stroma, approximating 40% of all cells of the body. Whereas, precursor cells are composed of cells that self-replicate and repair and/or replace worn out differentiated cells. Examples of these precursor cells are mesenchymal stem cells (MSCs) [3-12], very-small embryonic-like stem cells (VSELs) [13-18], multilineage-differentiating stress-enduring cells (MUSEs) [19-27], marrow-isolated adult multilineage inducible cells (MIAMIs) [28,29], small mobile stem cells (SMS) [30,31], and multipotent adult progenitor cells (MAPCs) [32-36].

In 1975, an extremely rare population of endogenous adult telomerase positive cells was discovered. These telomerase positive cells were located in multiple postnatal individuals [58-60] as quiescent hibernating cells within the connective tissue interstitium, and only following trauma would become activated, proliferate, migrate to the site of tissue damage and repair the damage [58].  Since 1975, these endogenous adult telomerase positive cells have been extensively characterized [61]. They have been identified in multiple species, isolated, sorted, cloned from single cells, genomically-labeled, expanded exponentially, functions examined with biological agents, long term cryopreservation [62-68], used in pre-clinical animal models of disease [69-73], and fresh isolates of autologous [74-82] and gender-matched ABO blood group-matched allogeneic cells [83-88] used in clinical studies of chronic disease, including from individuals in the ninth decade of their lives [88-90]. The current terminology for these endogenous adult cells is “adult telomerase positive stem cells”, for the presence of the telomerase enzyme, their ability for unlimited self-replication, their ability to form every cell type within the body, and their ability to repair and replace damaged cell, tissues, and organs. Utilizing adult telomerase positive stem cells, autologous and/or gender-matched ABO blood group-matched (or O-negative) allogeneic cells, reverse the signs and symptoms of their respective disease, restoring functionality to the individual [89,90].

Recently, a series of articles have been published detailing comparison and contrast methodologies used to work with these adult telomerase positive stem cells versus telomerase negative progenitor mesenchymal stem cells and other telomerase negative progenitor stem cells [62-68]. Unfortunately, early on it was noted that procedures for isolation and subsequent culturing differed significantly between adult telomerase positive stem cells and the telomerase negative progenitor cells [62-68]. In this study, the adult telomerase positive stem cells were tested to confirm or deny the assertion that ALL adult stem cells, be they telomerase negative progenitor cells or telomerase positive stem cells, decrease in number with increasing age of an individual. 

Materials and Methods

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).

Skeletal muscle biopsy specimens from newborn, pubescent (10-12-year-olds), sexually-mature (30-year-old), and geriatric (85-year-old) human [Homo sapiens] individuals were obtained from NIH/NIRS. Five-ml of Dulbecco’s Phosphate Buffered Saline, pH 7.4 (DBPS, GIBCO, Grand Island, NY) was added to each of 12 fifty-ml centrifuge tubes (Falcon, Thermo-Fisher Scientific, Waltham, MA), and labeled according, experiment number (#s: 1, 2, 3), age of  biopsied individual (newborn < 1-year-old [NB], pubescent 10-12-year-old [Pub], sexually mature 30-year-old [SM], and geriatric 85-year-old [Ger]), and enzyme mixture to be used (trypsin [T], collagenase-dispase [CD]), and tare weights taken.

The approximately 10-g of each biopsy specimen was individually placed into separate sterile 100-mm glass petri dishes in DPBS. Each biopsy specimen was macerated to the consistency of orange marmalade and separated into thirds. Each third was placed into separate pre-tared tubes. Final tube designations were as follows, e.g., #1-NB-T, #2-NB-CD, #3-NB-CD; #1-Pub-T, #2-Pub-CD, #3-Pub-CD; #1-SM-T, #2-SM-CD, #3-SM-CD; #1-Ger-T, #2-Ger-CD, and #3-Ger-CD. All tubes were placed on wet ice or at 4⁰C until further processing.

Experiment #1: Short term trypsin digest [37,91,92], as 10% (w/v) Trypsin (Sigma) in DPBS for 15 min.  Volume in tubes were measured, subtracted from 20, and resultant volume of 37^oC pre-warmed enzymatic solution (20% trypsin) added to each tube for a final concentration of 10% trypsin. The tubes were capped, sealed with Parafilm (Diagger, Buffalo Grove, IL), and placed into a weighted holder in a 37^oC shaker water bath (Thermo-Fisher Scientific) for 15 min. The tubes were removed from the water bath, Parafilm removed, and 20-ml of ice cold 1% collagen solution [63] added to each tube to stop protease/enzymatic process. The tubes were then centrifuged at 2,000 RCF (Relative Centrifugation Force) (centrifuge, Thermo-Fisher Scientific) to separate cell pellet from enzyme solution.

Following centrifugation, the supernatant was removed to bleach (Diagger), and cell pellet reconstituted in sterile DPBS. One hundred microliters of cell suspension were placed into 1.5-ml polypropylene uncapped microcentrifuge tubes (Corning, Corning, NY) using a P-200 Ranin micropipettor (Mettler Toledo, Columbus, OH). One hundred microliters of 0.4% Trypan blue (Kodak, Rochester, NY) in DPBS buffer [63] were added to the 1.5-ml polypropylene tubes containing the cell suspensions. The microcentrifuge tubes were capped, vortexed at 1-sec x 10, and returned to their holders. Each microcentrifuge tube was uncapped, 50 microliters of cell suspension/Trypan blue solution was removed, and placed onto each groove of a hemocytometer (Thermo-Fisher, Scientific). Using a NIKON TMS Brightfield/Phase Contrast microscope, samples were viewed at 10x magnification with brightfield optics. Cells were scored according to Figure 1, based on size and Trypan blue staining characteristics.

Experiment #2: Collagenase/Dispase digest for 15 min.  Volume in tubes were measured, subtracted from 20, and resultant volume of 37^oC pre-warmed enzymatic solution were added to each tube to a final concentration of 250 units/ml type-1 collagenase (Worthington Biochemicals, Lakewood, NJ) and 33.3 units/ml dispase (R&D Laboratories, Minneapolis, MN) in calcium-free, magnesium-free DPBS containing 25 mM ethylene glycol bis (b-aminoethyl ether)-N,N,N’,N’- tetraacetic acid (EGTA, Sigma) (25 mM = 0.475 g EGTA/500 ml calcium-/magnesium-free PBS), pH 7.4, was used to separate precursor cells from their extracellular matrices [63]. The tubes were capped, sealed with Parafilm, and placed in a weighted holder in a 37^oC shaker water bath for 15 min.

The tubes were removed from the water bath, Parafilm removed, and 20-ml of ice cold 1% collagen solution [63] added to each tube to stop enzymatic process. The tubes were then centrifuged at 2,000 RCF to separate cell pellet from enzyme solution. Following centrifugation, the supernatant was removed to bleach, and cell pellet reconstituted in sterile DPBS. One hundred microliters of cell suspension were placed into 1.5-ml polypropylene microcentrifuge tubes using a P-200 Ranin micropipettor. One hundred microliters of 0.4% Trypan blue in DPBS buffer were added to the 1.5-ml polypropylene capped tubes containing the cell suspensions.

The microcentrifuge tubes were capped, vortexed at 1-sec x 10, and returned to their holders. A microcentrifuge tube was uncapped, 50 microliters of cell suspension/Trypan blue solution was removed, and placed onto each groove of a hemocytometer (Thermo-Fisher, Scientific). Using a NIKON TMS Brightfield/Phase Contrast microscope, samples were viewed at 10x magnification with brightfield optics. Cells were scored according to Figure 1, based on size and Trypan blue staining characteristics.

Experiment #3: Samples were digested with type-1 collagenase/dispase for 15-min (NB, newborn), 1-hr. (Pub, 10-12-year-old), 3-hr. (SM, 30-year-old), and 8.5 hr. (Ger, 85-year-old). Volume in tubes were measured, subtracted from 20, and resultant volume of 37^oC pre-warmed enzymatic solution was added to each tube to a final concentration of 250 units/ml type-1 collagenase (Worthington Biochemicals, Lakewood, NJ) and 33.3 units/ml dispase (R&D Laboratories, Minneapolis, MN) in calcium-free, magnesium-free DPBS containing 25 mM ethylene glycol bis (b-aminoethyl ether)-N,N,N’,N’- tetraacetic acid (EGTA, Sigma) (25 mM = 0.475 g EGTA/500 ml calcium-/magnesium-free PBS), pH 7.4, was used to separate precursor cells from their extracellular matrices [63].

The tubes were capped, sealed with Parafilm, and placed in a weighted holder in a 37^oC shaker water bath for 15 min. The tubes were removed from the water bath, Parafilm removed, and 20-ml of ice cold 1% collagen solution added to each tube to stop enzymatic process. The tubes were then centrifuged at 2,000 RCF to separate cell pellet from enzyme solution. Following centrifugation, the supernatant was removed to bleach, and cell pellet reconstituted in sterile DPBS.

One hundred microliters of cell suspension were placed into 1.5-ml polypropylene microcentrifuge tubes using a P-200 Ranin micropipettor. One hundred microliters of 0.4% Trypan blue in DPBS buffer were added to the 1.5-ml polypropylene tubes containing the cell suspensions. The microcentrifuge tubes were capped, vortexed at 1-sec x 10, and returned to their holders. A microcentrifuge tube was uncapped, 50 microliters of cell suspension/Trypan blue solution was removed, and placed onto each groove of a hemocytometer (Thermo-Fisher, Scientific). Using a NIKON TMS Brightfield/Phase Contrast microscope, samples were viewed at 10x magnification with brightfield optics. Cells were scored according to Figure 1, based on size and Trypan blue staining characteristics.

Results

• Experiment #1: 15-min digestion with Trypsin: Trypan blue negative cells approximating 15 microns or larger were present in similar numbers (based on per gram tissue starting material) in newborn, pubescent, and sexually mature individuals, but declined in number in the geriatric-aged tissue samples. However, the remaining material in all the samples consisted of cell debris (bits and pieces Trypan blue-positive material with sharp edges and no discernible Trypan blue negative spherical cells). 
• Experiment #2: 15-min digestion with collagenase/dispase: Trypan blue negative cells approximating 15 microns or larger were present in similar numbers (based on per gram tissue starting material) in newborn, pubescent, and sexually mature individuals, but had declined in number in the geriatric-aged tissue samples. Trypan blue negative, negative/positive, and positive cells approximating 12 microns or smaller were present at similar levels to themselves in all four populations examined, e.g., newborn, pubescent, sexually mature, and geriatric-aged individuals, but were considerably fewer in number than the Trypan blue negative cells that were 15-microns or larger.
• Experiment #3: Digestion times increased dependent on age of the individual, e.g., 15-min (newborn), 1-hr. (pubescent), 3-hr. (sexually-mature), and 8.5-hr. (geriatric), with collagenase/dispase:  All cells, both Trypan blue negative cells approximating 15 microns or larger, and cells approximating 12-mincrons or less that were Trypan blue negative, negative/positive, and positive, showed about a two-fold increase in the numbers of released cells at all four digestion times examined. However, cells approximating 15-microns or larger still showed a decrease in number of cells in geriatric-aged individuals compared to newborn, pubescent, and sexually mature, while cells approximating 12-microns or less that were Trypan blue negative, negative/positive, and positive, remained constant with respect to themselves at all time points analyzed, but were still considerably fewer in number than the Trypan blue negative cells that were 15-microns or larger.

Discussion & Conclusions

In this study, the precursor cells were examined to confirm or deny the assertion that ALL adult stem cells, be they telomerase negative progenitor cells or telomerase positive stem cells, decrease in number with increasing age of an individual.  Multiple fresh skeletal muscle human [Homo sapiens] biopsy specimens (newborn [<1-year-old], puberty [10-12-yer-old], sexually mature [30-32-year-old], and geriatric aged [85-year-old] individuals) were obtained from NIH/NIRS. In the first experiment, the muscle biopsy specimens were digested for 15-minutes with trypsin at 37^oC [37,91,92], followed by processing for cell counting using Trypan blue exclusion. Using Figure 1 to distinguish telomerase negative progenitor cells from adult telomerase positive stem cells [62] was based both on Trypan blue staining patterns and size approximations. Telomerase negative progenitor cells were Trypan blue negative cells approximating 15 microns or larger and present in similar numbers (based on per gram tissue starting material) in newborn, pubescent, and sexually mature individuals, but declined in number in the geriatric-aged tissue samples. However, the remaining material in all the samples consisted of cell debris (bits and pieces Trypan blue-positive material with sharp edges and no discernible Trypan blue negative spherical cells). 

The theory for using trypsin to release cells from their extracellular matrices is that trypsin cleaves between adjacent lysine and arginine residues [93]. Thus, trypsin will disrupt the core proteins of glycoproteins and proteoglycans within the extracellular matrices, releasing the cells. Apparently, the cell membranes of adult telomerase positive cells also contain an abundance of adjacent lysine and arginine residues [93,94], whereby the trypsin digest caused loss of cell membrane integrity with resultant formation of cell debris.

In the second experiment, instead of using short term trypsin digest, a combination of enzymes was used that would disrupt the cells attachments to their connective tissue collagen substrata, e.g., type-1 collagenase and dispase, rather than to disrupt the cell plasma membranes themselves [63]. Multiple fresh skeletal muscle human [Homo sapiens] biopsy specimens (newborn, puberty, sexually mature, and geriatric aged individuals) were obtained from NIH/NIRS. The muscle biopsy specimens were macerated to the consistency of orange marmalade and then digested for 15-minutes with 250 Units of type-1 collagenase and 33.3 units of dispase at 37oC in DPBS containing EGTA [63], followed by processing for cell counting using 0.4% Trypan blue exclusion. 

Instead of using trypsin, 25 mM EGTA in calcium/magnesium-free buffer and two enzymes, collagenase and dispase, were used to release viable cells from their substratum. Cells within an individual are attached to their extracellular matrices through glycoprotein moieties in their basement membranes [95-97]. The basement membrane is a non-cellular structure which contains type-4 collagen, heparan sulfate proteoglycan, laminin, fibronectin, etc. [95,97]. Basement membrane fibronectin connects to type-12 collagen which is the bridge molecule to type-1 collagen, the predominant extracellular matrix glycoprotein in connective tissues [95,97]. The telomerase positive cells are attached to their collagen-based connective tissue substratum by two binding sites, a calcium-dependent binding site and an RGD-fibronectin-collagen dependent binding site [97]. 

EGTA is a specific calcium chelator, that has a higher binding coefficient than the calcium-dependent cell binding site [93], thus releasing the cells at this binding site. Collagenase and dispase disrupt the RGD-fibronectin-collagen binding site, releasing the cells from this binding site. By disrupting the extracellular matrix binding sites, rather than the cell membranes, intact viable cells are released from the connective tissues [98-102]. This was noted in the ability to recognize Trypan blue negative, negative/positive, and positive spherical entities of 12-microns or less in size [62].

Using Figure 1 to distinguish telomerase negative progenitor cells from adult telomerase positive stem cells based on Trypan blue staining patterns and size approximations, Trypan blue negative cells approximating 15 microns or larger were present in similar numbers (based on per gram tissue starting material) in newborn, pubescent, and sexually mature individuals, but had declined in number in the geriatric-aged tissue samples. Trypan blue negative, negative/positive, and positive cells approximating 12 microns of less were present at similar levels in all four populations examined, e.g., newborn, pubescent, sexually mature, and geriatric-aged individuals. However, the numbers of adult telomerase positive stem cells were significantly less than those of the progenitor cells at each time point examined.

Figure 1: Trypan Blue and CEA-CAM-1 Staining Patterns of a TPSCs and MSCs.

Acknowledgements

The author would like to thank JA F-C-Coleman, GF L-Black, NL Henson, J-I Yoon, for technical assistance; and Dr AC Black Jr for editorial assistance.  

Funding

Funding for this work was supported by research grants from Rubye Ryle Smith Charitable Trust, The MEDCEN Community Health Foundation of Central Georgia, Dragonfly Foundation for Research and Development, and Morphogen Pharmaceuticals, Inc.

Competing and conflicting interests

Endogenous adult telomerase positive stem cells (aTPSCs) have competing interests with those laboratories studying mixed populations of telomerase negative progenitor cells termed stem cells, e.g., mesenchymal stem cells (MSCs), very small-embryonic-like stem cells (VSELs), multilineage-differentiating stress-enduring cells (MUSEs), marrow-isolated adult multilineage inducible cells (MIAMIs), small mobile stem cells (SMSs), and multipotent adult progenitor cells (MAPCs). The conflicting interest relates to comparing innate cellular characteristics, putative differentiation potentials and ability to affect a positive regenerative response when transplanted in vivo of single cell-derived clones of aTPSCs versus mixed cell populations of MSCs, VSELs, MUSEs, MIAMIs, SMSs, and MAPCs.

NOMENCLATURE

Humans, Homo sapiens”

References

1. Young HE. (2026) Adult Telomerase Positive Stem Cells: Induced Proliferation of Precursor Cells by Platelet-Derived Growth Factor-BB. J Stem Cell Res. 7(1):1-15.
2. Young HE. (2025) Adult Telomerase Positive Stem Cells: Compare and Contrast Biobanking with Mesenchymal Stem Cells and Other Progenitor Cells. GSC Adv Res Rev. 25(03): 218-26.
3. Friedenstein AJ, Deriglasova UF, Kulagina NN,   Panasuk AF, Rudakowa SF,et al. (1974) Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol. 2:83-92.
4. Da Silva ML, Chagastelles PC, Nardi NB.(2006)  Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci.  119:2204-13.
5. Caplan AI. (1991) Mesenchymal stem cells. J Orthop Res. 9:641-50.
6. Caplan AI. (2005) Review: Mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng.   11:1198-211.
7. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas, R et al. (1999) Multilineages potential of adult human mesenchymal stem cells. Science. 284:143-47.
8. Caplan AI. (2017) Mesenchymal stem cells: time to change the name. Stem Cells Translational Medicine. 6(6): 1445-51. 
9. Caplan AI, Dennis JE. (2006) Mesenchymal stem cells as trophic mediators. J Cell Biochemistry. 98(5): 1076-84.
10. Li Z, Hu X, Zhong JF. (2019) Mesenchymal stem cells: characteristics, function, and application. Stem Cells Intl. 6: 8106818.
11. Via AG, Frizziero A, Oliva F. (2012) Biological properties of mesenchymal stem cells from different sources. Muscles Ligaments Tendons J. 2(3):154-62.
12. Caplan AI. (2009) Why are MSCs therapeutic? New data: new insight. J Pathol. 217(2): 318-24.
13. Ratajczak MZ, Ratajczak J, Kucia M. (2019) Very small embryonic-like stem cells (VSELs) – an update and future directions. Circ Res. 124(2): 208-10.
14. Zuba-Suma EK, Wojakowski W, Ratajczak MZ, Dawn B. (2011) Very small embryonic-like stem cells: biology and therapeutic potential for heart repair. Antioxid Redox Signal. 15(7):1821-34.
15. Ratajczak MZ, Zuba-Suma E, Wojakowski W, Kucia M.  (2014) Very small embryonic-like stem cells (VSELs) represent a real change in stem cell biology: recent pros and cons in the midst of a lively debate. Leukemia. 28: 473-84.
16. Danova-Alt R, Heider A, Egger D, Cross M, Alt R. (2012) Very small embryonic-like stem cells purified from umbilical cord blood lack stem cell characteristics. PLoS ONE. 7(4): e34899.
17. Ratajczak MZ, Shin D-M, Lui R, Mierzejewska K, Ratajczak J, et al. (2012) Very small embryonic/epiblast-like stem cells (VSELs) and their potential role in aging and organ rejuvenation – an update and comparison to other primitive small stem cells isolated from adult tissues. Aging 4(4):235-46.
18. Thetchinamoorthy K, Jarczak J, Kieszek P, Wierzbicka D, Ratajczak J, et al. (2015) Very small embryonic-like stem cells (VSELs) on the way for potential applications in regenerative medicine. Front Bioeng Biotechnol. 13:1564964.
19. Wakao S, Kitada M, Kuroda Y, Dezawa M. (2011) Multilineage-differentiating stress enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proc Natl Acad Sci USA. 108(24):9875-80.
20. Ossanna R, Veronese S, Sierra LAQ, Conti A, Conti G, et al. (2023) Multilineage-differentiating stress enduring (Muse) cells: an easily accessible, pluripotent stem cell niche and powerful properties for regenerative medicine applications. Biomedicines. 11(6): 1587.
21. Que H, Mai E, Hu Y, Li H, Zheng W, et al. (2024) Multilineage-differentiating stress enduring (Muse) cells: a powerful tool for tissue repair. Front Cell Dev Biol. 12:1380785.
22. Dezawa M. (2018) The Muse cell discovery, thanks to wine and science. Adv Exp Med Biol. 1103;1-11.
23. Alanazi RF, Alhwity BS, Almahlawi RM, Alatawi BD, Albalawi SA, et al. (2023) Multilineage-differentiating stress enduring (Muse) cells: a new era of stem cell-based therapy. Cells. 12(13):1676.
24. Yamamoto S, Shiraishi K, Kushida Y, Oguma Y, Wakao S, et al. (2025) Nose-to-brain delivery of human muse cells enhances structural and functional recovery in the murine ischemic stroke model. Sci Rep. 15:16243.
25. Velasco MG, Satue K, Chicharro D, Martins E, Rubio M. (2023) Multilineage-differentiating stress enduring (Muse) cells: the future of human and veterinary medicine. Biomedicines. 11(2):636.
26. Minatoguchi S, Fujita Y, Niizuma K, Tominaga T, Yamashita T, et al. (2024) Donor muse cell treatment without HLA-matching tests and immunosuppressant treatment. Stem Cells Transl Med. 13(6): 527-45.
27. Dezawa M. (2025) Macrophage- and pluripotent-like reparative muse cells are unique endogenous stem cells distinct from other somatic stem cells. Front Bioeng Biotechnol. 13: 1553382.
28. Grau-Monge C, Delcroix GJ-R, Bonnin-Marwuez A, Valdes M, D’lppolito GD. (2017) MIAMI cells embedded within a biologically-inspired construct promote recovery in mouse model of peripheral vascular disease. Biome Mater. 12(1):015024.
29. D’lppolito G, Diabira S, Howard GA, Menei P, Roos BA, et al. (2004) Marrow isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci. 117(Pt 14): 2971-81.
30. Smsbiotech.com
31. Rahmo A, Schechter R. (2024) Small Mobile Stem Cells, Unique Regenerative Effects on Lung of Emphysema Animal Model Currently Undergoing Phase 1 Clinical Trial. Am J Respir Crit Care Med. 209: A6431.
32. Williams R. (2007) Cell replacement therapy – are MAPCs the answer? J Exp Med. 204(1): 4.
33. Subramanian K, Geraerts M, Pauwelyn KA, Park D, Owens J. (2010) Isolation procedures and characterization of multipotent adult progenitor cells from rat bone marrow. Cell Program Reprogram Meth Prot. 55-78.
34. Reyes M, Verfaillie CM. (2001) Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal progenitor cells. Ann NY Acad Sci. 2938:231-33.
35. Verfaillie CM. (2005) Multipotent adult progenitor cells: an update. Hematol Am Soc Hematol Edu Program, 265: 55-65.
36. Jiang Y, Breyer A, Lien L, Blackstad M, Verfaillie C. (2004) Culture of multipotent adult progenitor cells (MAPCs). Blood. 104(11): 2329.
37. Caplan AI. (1994) The mesengenic process. Clint Plast Surg. 21:429-35.
38. Ahmed AS, Sheng MH, Wasnik S, Baylink DJ, Lau KW. (2017) Effect of aging on stem cells. World J Exp Med. 7(1):1-10.
39. Oh J, Lee YD, Wagers AJ. (2014) Stem cell aging: mechanisms, regulators and therapeutic opportunities. Nat Med. 20:870–80. 
40. Schultz MB, Sinclair DA. (2014) When stem cells grow old: phenotypes and mechanisms of stem cell aging. Development. 143:3–14.
41. Sousounis K, Baddour JA, Tsonis PA. (2014) Aging and regeneration in vertebrates. Curr Top Dev Biol. 108:217-46.
42. Wagner W, Bork S, Horn P, Krunic D, Walenda T, et al. (2009) Huber FX, Eckstein V, et al. Aging and replicative senescence have related effects on human stem and progenitor cells. PLoS One. 4:e5846. 
43. Liu H, Xia X, Li B. (2015) Mesenchymal stem cell aging: Mechanisms and influences on skeletal and non-skeletal tissues. Exp Biol Med (Maywood) 240:1099–106.
44. Sethe S, Scutt A, Stolzing A. (2005) Aging of mesenchymal stem cells. Ageing Res Rev. 5:91–116.
45. Stolzing A, Jones E, McGonagle D, Scutt A. (2008) Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev.129:163–73.
46. Fraile M, Eiro N, Costa LA, Martín A, Vizoso FJ. (2022) Aging and Mesenchymal Stem Cells: Basic Concepts, Challenges and Strategies. Biology (Basel). 11(11):1678.
47. Jiang X, Li W, Ge L, Lu M. (2023) Mesenchymal Stem Cell Senescence during Aging: From Mechanisms to Rejuvenation Strategies. Aging Dis. 14(5):1651-76.
48. Raggi C, Berardi AC. (2012) Mesenchymal stem cells, aging and regenerative medicine. Muscles Ligaments Tendons J. 2(3):239-42.
49. Fafián-Labora JA, Morente-López M, Arufe MC. (2019) Effect of aging on behaviour of mesenchymal stem cells. World J Stem Cells. 11(6):337-46.
50. Efimenko A, Dzhoyashvili N, Kalinina N, Kochegura T, Akchurin R. (2014)  Adipose-Derived Mesenchymal Stromal Cells From Aged Patients With Coronary Artery Disease Keep Mesenchymal Stromal Cell Properties but Exhibit Characteristics of Aging and Have Impaired Angiogenic Potential. Stem Cells Transl Med. 3(1): 32-41.
51. Meng Q, Zheng Y, Chen X, Mu J, Li C, et al. (2025) Mesenchymal Stem Cells Senescence: Mechanism and Rejuvenation Interventions. Int J Med Sci. 22(14):3692-708.
52. Ganguly P, El-Jawhari JJ, Burska AN, Ponchel F, Giannoudis PV, et al. (2019) The Analysis of In Vivo Aging in Human Bone Marrow Mesenchymal Stromal Cells Using Colony-Forming Unit-Fibroblast Assay and the CD45^lowCD271⁺ Phenotype. Stem Cells Int. 2019:5197983.
53. Brack AS, Muñoz-Cánoves P. (2016) The ins and outs of muscle stem cell aging. Skeletal Muscle. 6:1. 
54. García-Prat L, Sousa-Victor P, Muñoz-Cánoves P. (2013) Functional dysregulation of stem cells during aging: a focus on skeletal muscle stem cells. FEBS J. 280:4051-62. 
55. Gonen O, Toledano H. (2014) Why adult stem cell functionality declines with age? Studies from the fruit fly Drosophila melanogaster model organism. Curr Genomics. 15(3):231-6.
56. Narbonne P. (2018) The effect of age on stem cell function and utility for therapy. Cell Med. 10:2155179018773756.
57. Schultz MB, Sinclair DA. (2016) When stem cells grow old: phenotypes and mechanisms of stem cell aging. Development. 143(1):3-14.
58. Young HE. (2004) Existence of reserve quiescent stem cells in adults, from amphibians to humans. Curr Top Microbiol Immunol. 71-109.
59. Young HE. (2025) Carcinoembryonic antigen-cell adhesion molecule-1 and stage-specific embryonic antigen-4 are present in the reproductive organs of adult mammals. GSC Adv Res Rev. 23(03): 149-57.
60. Young HE. (2025) Totipotent stem cells and pluripotent stem cells are present in the reproductive organs of an adult mammal. GSC Adv Res Rev. 23(03):158-80. 
61. Young HE, Speight MO. (2020) Characterization of endogenous telomerase-positive stem cells for regenerative medicine, a review. Stem Cell Regen Med. 4(2):1-14.
62. Young HE. (2025) Adult telomerase positive stem cells: introduction and location. GSC Advanced Research and Reviews.  25(02): 296-331.
63. Young HE. (2025) Adult telomerase positive stem cells: isolation, plating, and propagation. GSC Advanced Research and Reviews. 25(02):407-40.  
64. Young HE. (2025) Adult telomerase positive stem cells: differential cryopreservation, cell surface marker profiles, and cell sorting. MOJ Orthop Regen. 17(5): 155-73.
65. Young HE. (2025) Adult telomerase positive stem cells: Cell-Specific Exosome-Conditioned Medium, Repetitive Single Cell Clonogenic Analysis, and Genomic Labeling. GSC Adv Res Rev. 25(2): 480-504.
66. Young HE. (2025) Adult telomerase positive stem cells: Effects of Biological Agents on Single Cell Clones of Adult Cells. GSC Adv Res Rev. 25(03):028-47. 
67. Young HE. (2025) Adult Telomerase Positive Stem Cells: Effects of Biological Agents on Genomically-Labeled Clones and Unlabeled Clones of Adult Cells. GSC Adv Res Rev. 25(03): 127-45.
68. Young HE. (2025)   Adult Telomerase Positive Stem Cells: Compare and Contrast Biobanking with Mesenchymal Stem Cells and Other Progenitor Cells. GSC Adv Res Rev. 25(03): 218-26.
69. Young HE, Duplaa C, Yost MJ, Henson NL, Floyd JA, et al. (2004) Clonogenic analysis reveals reserve stem cells in postnatal mammals. II. Pluripotent epiblastic-like stem cells. Anat. Rec. 277A:178-203.
70. Young HE, Hyer L, Black AC Jr, Robinson Jr JS. (2013) Adult stem cells: from bench-top to bedside. In: Tissue Regeneration: Where Nanostructure Meets Biology, 3DBiotech, North Brunswick, NJ 2013; Chap 1, pp 1-60,
71. Young HE, Limnios IJ, Lochner F, McCommon G, Black GF, et al. (2017) cardiovascular disease and adult healing cells: From bench top to bedside. J Stem Cell Res. 1(3) 002:1-8.
72. Young HE, Black GF, Coleman JA, Hawkins KC, Black Jr AC. (2017) pulmonary diseases and adult healing cells: from bench top to bedside. J Stem Cell Res.1(2) 003:1-9.
73. Young HE, Limnios JI, Lochner F, McCommon G, Cope LA, et al. (2017) Pancreatic islet composites secrete insulin in response to a glucose challenge. J Stem Cell Res. 1(1) 001: 1-12.
74. Young HE, Speight MO. (2020) Informed consent guidelines for optimizing the use of telomerase-positive stem cells for regenerative medicine. J Regen Med Biol Res.1(1):1-20.
75. Young HE, Hyer L, Black AC Jr, Robinson Jr JS. (2013) Treating Parkinson disease with adult stem cells. J Neurological Disorders. 2:107-109.
76. Young HE, Speight MO. (2020) Osteoarthritis Treated with Telomerase-Positive Adult Stem Cells in Animals and Humans. Stem Cells Regen Med. 2020; 4(2):1-11.
77. Young HE, Speight MO. (2020) cardiovascular disease treated with telomerase-positive stem cells. Stem Cells Regen Med. 4(2):1-8.
78. Young HE, Speight MO. (2020) Age-related [dry] macular degeneration treated with autologous telomerase-positive totipotent stem cells. Stem Cells Regen Med. 4(3):1-9.
79. Young HE, Speight MO. (2021) Blunt force trauma-induced total bilateral vision impairment of 13 years duration treated with autologous telomerase positive stem cells. Stem Cells Regen Med. 5(1):1-22.
80. Young HE, Speight MO. (2021) Traumatic spinal cord injury treated with autologous telomerase-positive stem cells. Stem Cells Regen Med. 5(1):1-13.
81. Young HE, Speight MO. (2021) Chronic inflammatory demyelinating polyneuropathy treated with autologous telomerase-positive stem cells. Stem Cells Regen Med. 5(2):1-16.
82. Young HE, Speight MO. (2021) Treating Parkinson Disease with Autologous Telomerase-Positive Stem Cells, Update 2021. Stem Cells Regen Med. 5(1): 1-13.
83. Young HE, Speight MO. (2020) Donor selection is a critical component using naïve endogenous adult stem cells for the treatment of chronic diseases and traumatic injuries. J Regen Med Biol Res. 1(2):1-28.
84. Young HE, Speight MO. (2020) Allogeneic and autologous telomerase-positive stem cells as a potential treatment for systemic lupus erythematosus. Stem Cells Regen Med.  4(2):1-9.
85. Young HE, Speight MO. (2020) Telomerase-positive stem cells as a potential treatment for idiopathic pulmonary fibrosis. Stem Cells Regen Med. 4(2):1-11.
86. Young HE, Speight MO. Potential treatment of chronic obstructive pulmonary disease with allogeneic and autologous telomerase-positive stem cells. Stem Cells Regen Med. 2020; 4(3):1-11.
87. Young HE, Speight MO. (2020) Allogeneic telomerase-positive stem cells as a treatment for celiac disease. Stem Cells Regen Med. 4(2):1-7.
88. Young HE, Speight MO. (2021) Alzheimer’s disease treated with autologous and allogeneic telomerase-positive stem cells. Stem Cells & Regen Med. 5(1):1-17.
89. Young HE. (2023) 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 Adv Res Rev. 16(1):066-81.
90. Young HE. (2024) Combinatorial Nutraceutical Supplement Pill (CNSP) Stimulates Naïve Adult Telomerase Positive Stem Cells In-Situ to Heal Cardiomyopathies. GSC Adv Res Rev. 20(02), 047-56.
91. ATCC Animal Cell Culture Guide, The Essentials of Life Sciences Research, American Type Culture Collection, Gaithersburg, Maryland.
92. www.worthington-biochem.com Introduction to Enzymes.
93. Lieberman M, Marks AD. Mark’s Basic Medical Biochemistry, A Clinical Approach, 4^th Edition. Lippincott Williams & Wilkins.
94. Kierszenbaum AL. (2002) Histology and Cell Biology, An Introduction to Pathology. Elsevier/Mosby, 11830 Westline Industrial Dr, St Louis, MO 63146. ISBN: 0-323-01639-1.
95. Young HE, Carrino DA, Caplan AI. (1989) Histochemical analysis of newly synthesized and resident sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg. J Morphol. 201:85-103.
96. Young HE, Carrino DA, Caplan AI. (1990) Changes in synthesis of sulfated glycoconjugates during muscle development, maturation, and aging in embryonic to senescent CBF-1 mouse. Mechanisms of Ageing and Development. 53:179-93.
97. Yue B. (2014) Biology of the extracellular matrix. J Glaucoma. S20-23.
98. Young HE, Ceballos EM, Smith JC, Lucas PA, Morrison DC. (1992) Isolation of embryonic chick myosatellite and pluripotent stem cells. J Tissue Culture Methods. 14:85-92.
99. Young HE, Ceballos EM, Smith JC, Mancini ML, Wright RP, et al. Pluripotent mesenchymal stem cells reside within avian connective tissue matrices. In Vitro Cell Devel Bio. 29A:723-36.
100. Pate DW, Southerland SS, Grande DA, Young HE et al. Isolation and differentiation of mesenchymal stem cells from rabbit muscle. Surgical Forum. XLIV:587-89.
101. Young HE, Steele T, Bray RA, Hudson J, Floyd JA, et al. (2001) 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. 264:51-62.
102. Stout CL, Ashley DW, Morgan III JH, Long GF, Collins JA, et al. (2007) Primitive stem cells reside in adult swine skeletal muscle and are mobilized into the peripheral blood following trauma. Am Surg. 73 (11):1106-10.
103. Young HE, Carrino DA, Caplan AI. (1998) Initial characterization of small proteoglycans synthesized by embryonic chick leg muscle-associated connective tissues. Connective Tissue Research. 17:99-118.
104. Young HE, Young VE, Caplan AI. (1989) Comparison of fixatives for maximal retention of glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated radiolabeled [35S] sulfate. J Histoch Cytochemist. 37:223-28.
105. Young HE, Carrino DA, Caplan AI. (1989) Histochemical analysis of newly synthesized and resident sulfated glycosaminoglycans during Musculo genesis in the embryonic chick leg. J Morphol. 201:85-103.
106. Young HE, Carrino DA, Caplan AI. (1990) Changes in synthesis of sulfated glycoconjugates during muscle development, maturation, and aging in embryonic to senescent CBF-1 mouse. Mechanisms of Ageing and Development. 53:179-93.