Method for increasing circulating levels of mesenchymal stem cells via administration to the oro- nasopharyngeal tract

Abstract

Disclosed is a non-invasive method for elevating circulating levels of therapeutically effective mesenchymal stem cells (MSCs). The method comprises (i) selecting, isolating or enriching a sub-population of pluripotent, migratory MSCs co-expressing SSEA3, S1PR2 and CD105 and (ii) administering the enriched cells to the oro-nasopharyngeal tract as a spray, aerosol, liquid, gel, lozenge or patch. The enriched cells exhibit enhanced survival, transendothelial migration and engraftment, overcoming pulmonary sequestration that limits intravenous MSC delivery. Purity tiers of ≥ 10 %, 20 %, 50 %, 75 % and 90 % triple-positive cells allow dose tailoring, and formulations optionally containing cryoprotectants, pro-survival or absorption enhancers. Sources include bone marrow, adipose, or birth tissues. Continuous, intermittent or bolus dosing can be self-administered and is applicable to prophylaxis, systemic regeneration, inflammation modulation and tissue repair. The invention provides a patient-friendly and non-invasive method that safely increases circulating MSC numbers and improves therapeutic efficacy.

Description

Method for Increasing Circulating Levels of Mesenchymal Stem Cells via Administration to the Oro- Nasopharyngeal TractPRIOR ART

[0001] Y. Kuroda et al., "Unique multipotent stress-enduring cells in adult human mesenchymal cell populations," Proc. Natl. Acad. Sci. U.S.A., 2010.Describes isolation of SSEA-3+ Muse cells and their tail-vein infusion into immunodeficient mice with various tissue injuries. While many cells homed to damaged sites, a substantial fraction was sequestered in pulmonary and splenic capillaries, reducing the efficiency of delivery to target organs.

[0002] S. Wakao et al., "Regenerative Effects of Mesenchymal Stem Cells: Contribution of Muse Cells," PLoS One, 2014.Demonstrates intravenous administration of Muse cells in models of hepatitis, muscle degeneration, spinal cord injury and skin wounds. Reports that "some Muse cells were trapped in the lung and spleen," highlighting first-pass retention as a bottleneck for systemic therapy.

[0003] US 10,293,003 B2 (Dezawa et al.), "Multilineage-differentiating stress-enduring (Muse) cells for regenerative medicine," published 2019.Claims intravascular (systemic) infusion of Muse cells to treat chronic kidney disease, with accumulation in renal tissue. Does not address— or propose solutions for— the pulmonary- capillary entrapment that limits the fraction of cells reaching distant targets.

[0004] T. Abe et al., "Intravenously transplanted human Muse cells afford brain repair in a mouse lacunar stroke model," Stroke, 2020.Shows functional recovery after IV Muse cell infusion in stroke, but relies on models where lung filtration still removes a significant cell fraction before cerebral engraftment.BACKGROU ND OF THE INVENTION

[0005] The present invention relates to methods for enhancing systemic distribution and therapeutic efficacy of mesenchymal stem cells. Specifically, it pertains to increasing circulatinglevels of therapeutically effective mesenchymal stem cells (MSCs) by administering selected, isolated, purified or enriched populations of pluripotent, migratory MSCs, characterized by surface markers SSEA3+, S1PR2+, and CD105+, via non-invasive Oro-Nasopharyngeal administration. Additionally, various formulations are provided to aid enhanced effectiveness.

[0006] Mesenchymal stem cell (MSC) therapies hold significant therapeutic potential due to their regenerative capabilities, anti-inflammatory and immunomodulatory properties, and capacity for tissue repair through paracrine mechanisms as well as via limited engraftment and differentiation. However, conventional MSC therapies administered via conventional MSC administration methods, such as intravenous or direct injection, have significant limitations including cellular entrapment in pulmonary capillaries, limited systemic distribution, poor survival, poor engraftment, reduced therapeutic efficacy, patient discomfort, invasiveness, high costs, and clinical resource demands.

[0007] A clear need exists for improved MSC administration methods that enhance systemic distribution, survival, and therapeutic efficacy, providing superior therapeutic outcomes with minimal invasiveness and resource use.

[0008] Furthermore, current cell therapies administering SSEA3+, S1PR2+, and CD105+ MSCS do so in limited settings and with limited dosing regimen flexibility due to the invasive, costly, technical and resource intensive injection based administration methods. The inability to dose on-demand and frequently, hampers these potent cells potential for extensive and continuous regeneration. By novel combination of pluripotent, migratory SSEA3+, S1PR2+, and CD105+ MSC therapy, with oro-nasopharyngeal tract delivery, non-invasive, patient friendly, on-demand, and highly effective methods of treating and preventing patient health measures are afforded by this invention.SU MMARY OF THE INVENTION

[0009] The present invention addresses existing limitations by providing a method for increasing the circulating levels and systemic availability of therapeutically effective MSCs through the synergistic administration of Selected, isolated, purified or enriched pluripotent, migratory MSC populations, characterized specifically by surface markers SSEA3+, S1PR2+, and CD105+, via non-invasive Oro-Nasopharyngeal delivery.DETAI LED DESCRIPTION OF TH E I NVENTION

[0010] At least one specification heading is required. Please delete this heading section if it is not applicable to your application. For more information regarding the headings of the specification, please see MPEP 608.01(a).

[0011] Stem Cell Sourcing:

[0012] The method involves first obtaining a source of bulk Mesenchymal Stem Cells, characterized by the simple standards such that the bulk MSC population should be plastic adherent under standard culture conditions, express CD73, CD90, and CD105, and lack expression of CD45, CD34, and HLA-DR.

[0013] These MSCs can be obtained from any suitable human tissue source or cell culture, such as, but not limited to, from skin, bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, placenta, placental blood, peripheral blood, amniotic tissue, amniotic fluid, transdifferentiated hematopoietic stem cells, trans-differentiated fibroblasts, trans-differentiated trophoblast stem cells, differentiated induced pluripotent stem cells or differentiated embryonic stem cells. Such tissue or cellular sources of MSCs can be obtained from live or post-mortem human sources, extra-embryonic sources, as well as from in-utero fetuses, as well as from fetal tissue obtained from aborted or ectopic pregnancies.

[0014] Selection, Isolation, Purification or Enrichment:

[0015] Such pluripotent and migratory MSC sub-populations are characterized by being positive under standard flow cytometry assays for the 3 surface markers: SSEA3, S1PR2, and CD105. These MSCs exhibit enhanced migratory capabilities, superior systemic biodistribution, and increased survival upon administration, thereby effectively increasing therapeutic MSC levels in circulation in the treated person.

[0016] Due to the limitations on cell density and total volume of administered cell formulations for Oro-Nasopharyngeal delivery, having a higher concentration of the cells delivered be the more effective cells provides an enhanced therapeutic outcome given the same number of total cells delivered. Furthermore, the mere existence of non-pluripotent MSCs in an intranasal delivery experiment has been shown to interfere with signaling crucial to the effectiveness of the pluripotent, migratory MSCs, when the concentration of the pluripotent, migratory MSCs is low, as it is naturally in bulk MSC populations (Yamamoto et al., 2025). Therefore theenrichment and purification of the pluripotent, migratory MSCs, which are then to be included in the final intranasal formulation and delivery, is crucial to the optimal effectiveness of this method.

[0017] In order to be able to select, isolate, purify or enrich the desired pluripotent, migratory MSCs, many suitable methods are available, which can be performed by a scientist skilled in the art, with the same result of increasing the concentration of pluripotent, migratory MSCs among the total population of MSCs. Such methods include but are not limited to: a. selective stress tolerance resistance based selection, such as with stressful culture of MSC populations in low oxygen environments under 5% 02, in serine protease enzyme containing media, in collagenase enzyme containing media, in serum-free low glucose media, in H202 containing or high ROS containing media, in cell death activating compound (e.g., cytotoxic antibody-drug conjugate, chemotherapeutic) containing culture, in suspension culture, in cold conditions, in freezing conditions, or in any combination of stressful conditions. b. antibody based selection, such as through the use of antibody-mediated cell sorting techniques known to one skilled in the art, including magnetic cell sorting (MACS), fluorescence-activated cell sorting (FACS), buoyancy-activated cell sorting (BACS), or combinations thereof.ln one preferred embodiment such method of selective stress tolerance resistance based selection comprises the exposure of bulk MSC populations to DM EM media containing sufficient trypsin concentrations (greater than or equal to 0.25% trypsin) for sufficient continuous time (greater than or equal to 48 hours) and collecting thereafter the surviving cells via centrifugation at 300xg rpm for 5 minutes, discarding the supernatant containing dead cells, seeding in adherent MSC conditions the remaining cell pellet, and 48 hours after discarding the supernatant seeded media and dead cells which have not survived and attached the to the cell culture surface, therein the process enriching for the pluripotent, migratory cells which can more selectively survive such culture conditions, achieving in such a process a concentration of greater than or equal to 10% pluripotent, migratory MSCs among the total resulting MSC population. In order to further enhance enrichment levels, such a process of trypsin treatment can be repeated sequentially on the surviving population or cultures derived thereof at any later time or immediately thereafter, thereby successively increasing the relative concentration of the desired pluripotent, migratory MSCs witheach trypsin treatment. Such a process may also be combined with other antibody based selection and selective stress tolerance resistance based selection methods aforementioned and known to one skilled in the art.

[0018] In another preferred embodiment a method to achieve a concentrated and purified pluripotent, migratory MSC population is to first sufficiently incubate, and therein tag amongst, a bulk MSC population with a suitable rat igM anti-SSEA3 antibody. Subsequently, the bulk MSC population is tagged further with sufficient secondary anti-rat igM magnetic beads. Thereafter, the magnetic bead bound ssea3+ cells are trapped in a suitable magnetic field suitable for cell isolation, such as within a commercially available MACS column. Thereafter such magnetically trapped cells are eluted and collected to obtain a purified target population of a SSEA3+, S1PR2+ and CD105+ cell population of equal to or greater than 10% concentration among the total collected MSC population. In order to further enhance purification levels, such a process of antibody mediated selection can be repeated sequentially on the collected target population or cultures derived thereof at any later time or immediately thereafter, thereby successively increasing the relative concentration of the desired pluripotent, migratory MSCs with each purification process. Such a process may also be combined with other antibody based selection and selective stress tolerance resistance based selection methods aforementioned and known to one skilled in the art.

[0019] Preferred embodiments of methods to enrich or purify the pluripotent, migratory MSC population more preferably result in at least about 20%, 50%, 75%, or 90% of MSCs expressing SSEA3, S1PR2, and CD105 simultaneously.

[0020] Selected, isolated, purified or enriched pluripotent, migratory MSCs can thereafter optionally be further cultured without reduction in concentration below 10% of the total MSC population, as can be periodically verified by standard flow cytometry assays. If such significant reduction in concentration of pluripotent, migratory MSCs below 10% occurs, or if a higher concentration of pluripotent, migratory MSCs is desired in any case, such methods of enrichment or purification may be performed again, once, or multiple times, alone, or in combination.

[0021] In order to verify the population of pluripotent, migratory MSCs with flow cytometry, cells are stained with anti-SSEA-3-FITC (clone MC-631, 1 pg / 10A6 cells), anti-SlPR2-PE (clone7F7, 0.5 pg / 10A6) and anti-CD105-APC (clone 266, 0.25 pg / 10A6). Single-cell events were gatedon FSC-A vs FSC-H, dead cells excluded with 7-AAD, and then triple-positive gates were set using FMO controls to <0.1 % spill-over.

[0022] Cryoprotectant and Supportive Formulation:

[0023] Selected, isolated, purified or enriched pluripotent, migratory MSCs are thereafter formulated in a pharmaceutically acceptable formulation, such as a cryoprotectant, delivery or carrier solution. In a preferred embodiment the cryoprotectant formulation is DMSO-free, and excipient grade. In another preferred embodiment the pharmaceutically acceptable formulation functions as both a cryoprotectant and delivery or carrier solution, and such dual functioning formulation is directly administered in-vivo along with the MSCs, comprising DMSO-free, excipient grade, sterile, molecules and carriers. In another preferred embodiment the cryoprotective formulation is later thawed after being frozen and reconstituted in a delivery solution.

[0024] Such DMSO-free active cryoprotectant agents may include, but are not limited to, organic osmolytes, organic polymers and albumin. In one embodiment this formulation comprises 5% trehalose, 5% recombinant human albumin and 90% lactated ringers solution — which avoids DMSO-associated toxicity and can serve directly as the final delivery carrier or solution, or as an intermediary solution for later reconstitution after freezing and thawing into a suitable final formulation. Due to the non-toxic DMSO-free nature of the formulation and ommitance of the requirement for washing away the DMSO prior to administration, less vital cells are lost during such wash steps, less equipment, time and cost is needed at the point of administration, and the viability and stability is increased, such that greater than 95% of postthawed cells are viable.

[0025] Delivery solutions can be simple conventional carriers such as sterile saline solutions, buffered solutions, and liposomal solutions, or formulated with additional components in order to further to enhance the administered MSCs survival, migration, and bioavailability, which can include: a. absorption-enhancing agents such as bile salts (e.g., sodium cholate 0.5 - 2.5 mg ml-1), surfactants (e.g., Tween 800.01 - 0.20 mg mL’1), or enzymes (MMP-9 1 - 50 pg mL’1, trypsin 0.01 - 1 mg mL-1, collagenase 0.01 - 0.30 mg mL-1, hyaluronidase 0.1 - 10 mg mL-1, bromelain 0.1 - 5 mg mL-1);b. bronchodilating or vasodilating agents such as 0-agonists (e.g., albuterol 0.3 - 1 mg mL-1), magnesium sulfate 1 - 12 mg mL-1, nitric-oxide-releasing agents (e.g., S- nitrosoglutathione 0.02 - 0.10 mg mL-1, NOSH-aspirin 0.4 - 8 pg mL-1), or epinephrine 10 - 200 pg mL-1; c. muco-adhesive polymers such as Carbopol® 980 NF 1 - 10 mg mL-1; d. extracellular-matrix proteins such as hyaluronic acid 0.5 - 10 mg mL-1; e. osmolytes such as trehalose 17 - 103 mg mL-1; f. cell-death inhibitors such as apoptosis inhibitors (e.g., Z-VAD-FMK 4.7 - 23 pg mL-1), pyroptosis inhibitors (e.g., VX-765 5 - 52 pg mL-1), anoikis inhibitors (e.g., Y-27632 0.34 - 6.8 pg mL-1), ferroptosis inhibitors (e.g., deferoxamine 5.6 - 56 pg mL-1), necroptosis inhibitors (e.g., Necrostatin-1 2.3 - 11 pg mL-1), necrosis inhibitors (e.g., Cyclosporin A 0.6 - 6 pg mL-1) and parthanatos inhibitors (e.g., PJ34 0.35 - 7 pg mL-1); g. cell-stress inhibitors such as integrated-stress-response inhibitors (e.g., ISRIB 45 - 450 ng mL-1), heat-shock proteins (e.g., HSP70 1 - 10 pg mL-1), cold-shock proteins (e.g., RBM3 0.1 - 1 pg mL-1), HERO proteins (e.g., SERBP1 / HERO45 0.1 - 1 pg mL-1), coldinducible RNA-binding proteins (e.g., CIRP 0.1 - 1 pg mL-1), endoplasmic-reticulum stress inhibitors (e.g., 4-phenylbutyric acid 0.08 - 0.82 mg mL-1), mitochondrial stress inhibitors (e.g., Elamipretide / SS-31 0.11 - 5.6 pg mL-1), cytoskeletal stress inhibitors (e.g., Blebbistatin 0.3 - 2.9 pg mL-1), JNK inhibitors (e.g., SP600125 0.22 - 4.4 pg mL-1), p38 inhibitors (e.g., SB203580 0.38 - 7.5 pg mL-1), p53 inhibitors (e.g., Pifithrin-a 3.4 - 13.5 pg mL-1), pl6 inhibitors (e.g., CDKN2A siRNA 0.13 - 0.65 pg mL-1), p21 inhibitors (e.g., UC2288 0.17 - 1.7 pg mL-1) or DNA-repair agents (e.g., OGGI activators 0.3 - 3 pg mL-1); h. antioxidants such as glutathione 0.15 - 1.5 mg mL-1; i. chemokine-receptor modulators such as SIP 0.04 - 3.8 pg mL-1; j. anti-inflammatory agents such as IL- 10 10 - 500 ng mL-1, Resolvin DI 4 - 188 ng mL-1or corticosteroids (e.g., fluticasone 25 - 250 ng mL-1); k. biologic agents, such as exosomes 109- 1011particles mL-10.1 - 10 pg total protein mL-1), platelets 1 x 10s- l x 107mL-1, mitochondria 109- 1011mL-1, peptides (e.g., Cu- GHK 0.4 - 40 pg mL-1), or growth factors (NGF 10 - 100 ng mL-1, BDNF 25 - 100 ng mL-1, FGF4 10 - 50 ng mL-1, WNT3a 50 - 200 ng mL-1);l. anti-clumping agents such as sodium heparin 1 - 100 IU mL-1, sodium citrate 1.5 - 7.3 mg mL-1and EDTA 0.19 - 1.86 mg mL-1; m. membrane protectants or repairing agents (e.g., Poloxamer 188 0.1 - 10 mg mL-1, a- tocopherol 4.3 - 43 pig mL-1); n. nuclear-envelope protectants or repairing agents (e.g., lonomycin 0.019 - 0.187 pig mL-1, Fusicoccin-A 0.05 - 2.4 pig mL-1); o. cytoskeletal-rearrangement enhancing agents (e.g., lysophosphatidic acid 0.43 - 4.3 pig mL-1, CN03 0.1 - 1 pg mL-1, phorbol 12-myristate 13-acetate 0.6 - 30 ng mL-1); p. phagocytosis-enhancing agents (e.g., GM-CSF 1 - 50 ng mL-1, monophosphoryl lipid A 10 - 1 000 ng mL-1, -1,3-glucan 10 - 500 pg mL-1, BML-1110.03 - 3.2 pg mL-1); q. senolytic agents (e.g., Dasatinib 0.05 - 0.5 pg mL-1, Quercetin 1.5 - 15 pg mL-1, Fisetin 2.9 - 14 pg mL-1, Navitoclax 0.10 - 0.97 pg mL-1); r. oncolytic agents (e.g., Etoposide 0.59 - 5.9 pg mL-1, Cisplatin 0.15 - 1.5 pg mL-1).

[0026] Synergistic Benefits of Supportive Formulations: a. Absorption-enhancing agents such as sodium cholate, Tween 80, and ECM-degrading enzymes (MMP-9, trypsin, collagenase, hyaluronidase, bromelain) transiently loosen mucus and intercellular junctions, enabling naso-oropharyngeally applied MSCs to traverse the epithelium and enter the vasculature more efficiently for systemic migration. b. Bronchodilators or vasodilators— p2-agonists (albuterol), magnesium sulfate, nitric-oxide donors (S-nitrosoglutathione, NOSH-aspirin), and epinephrine— expand airway caliber and increase mucosal perfusion, thereby boosting convective transport of MSCs into richly vascular submucosal beds for rapid dissemination. c. Mucoadhesive polymers such as Carbopol 980 NF form a bioadhesive hydrogel on the mucosa that lengthens residence time, minimizes formulation drip-out, and maintains intimate contact between MSCs and the absorbent epithelium. d. Extracellular-matrix components like hyaluronic acid provide a viscoelastic, CD44- binding scaffold that supports MSC viability, reduces anoikis, and guides directional migration toward chemotactic injury signals.e. Osmolytes such as trehalose stabilize lipid membranes and proteins against dehydration, osmotic shock, and shear during aerosolization and mucosal exposure, preserving MSC viability and secretory potency. f. Broad-spectrum cell-death inhibitors— including Z-VAD-FMK, VX-765, Y-27632, deferoxamine, necrostatin-1, cyclosporin A, and PJ34— protect MSCs from apoptosis, pyroptosis, anoikis, ferroptosis, necroptosis, necrosis, and parthanatos triggered by stresses of various delivery methods and in injured or degenerative tissue, prolonging functional persistence. g. Cell-stress modulators such as ISRIB, HSP70, RBM3, SERBP1 / HERO45, CIRP, 4- phenylbutyric acid, SS-31, blebbistatin, SP600125, SB203580, pif ithrin-a, CDKN2A siRNA, UC2288, and OGGI activators dampen translational, ER, mitochondrial, cytoskeletal, kinase, and DNA-damage stress pathways that would otherwise curtail MSC survival, engraftment and paracrine efficacy. h. Antioxidants like glutathione scavenge reactive oxygen and nitrogen species abundant in inflamed airway mucosa, preventing oxidative injury to MSC membranes, DNA, and secreted factors. i. Chemokine-receptor modulators such as sphingosine-l-phosphate (SIP) engage S1P- receptor gradients to enhance MSC chemotaxis and egress from mucosal tissue into vascular channels. j. Anti-inflammatory agents— including IL-10, Resolvin DI, and corticosteroids such as fluticasone— suppress local cytokine storms, lowering immune-mediated clearance of transplanted MSCs and fostering a pro-repair milieu. k. Biologies such as exosomes, platelets, donor mitochondria, bioactive peptides (Cu-GHK), and growth factors (NGF, BDNF, FGF4, WNT3a) co-stimulate MSCs and work in concert to release regenerative cues and accelerate more complex and complete tissue healing and regeneration. l. Anti-clumping agents like sodium heparin, sodium citrate, and EDTA chelate divalent cations and reduce cell-cell adhesion, maintaining a uniform MSC suspension that minimizes embolic risk and maximizes mucosal absorption area. m. Membrane-protective or repairing agents fortify or reseal phospholipid bilayers compromised by surfactants, enzymes, shear stress and various forces of variousdelivery methods such as aerosolization, sustaining MSC health, viability and efficacy after administration. n. Nuclear envelope protective or repairing agents helps mitigate the stresses of oro- nasopharyngeal delivered MSCs, which must squeeze through epithelial tight junctions, olfactory foramina, perivascular spaces and, eventually, micro-capillary basal laminae after intranasal dosing. Each micron-scale constriction can deform the nucleus enough to rupture the NE, exposing chromatin to cytosolic nucleases and cGAS, activating DNA- damage checkpoints, and triggering apoptosis or senescence. Nuclear envelope protective or repairing agents can activate ESCRT-lll-mediated sealing which normally patches these tears within minutes. o. Cytoskeletal-rearrangement enhancers activate actin-remodeling pathways that increase MSC deformability and transmigration through tight extracellular matrices toward zones of injury. p. Phagocytosis-enhancing agents stimulate host efferocytic clearance of debris, pathogens and non-viable, senescent or cancerous cells in the airway, reducing inflammatory barriers and allowing MSC immunomodulatory functions to predominate, as well as stimulate the phagocytic capacity of the administered MSCs themselves for efferocytic clearance of debris, pathogens and non-viable, senescent or cancerous cells. q. Senolytic agents selectively eliminate pro-inflammatory senescent cells in the host, provide migration promoting signaling and chemotaxis enablement, and provide a niche for the administered MSCs to engraft and replace the cleared senescent cells. r. Oncolytic agents eliminate cancer cells, mitigating the potential risk of the delivered MSCs promoting oncogenesis, as well as creating niche space where therapeutic MSCs can engraft, repair and exert regenerative effects.

[0027] Carriers and Delivery Formulation:

[0028] The pharmaceutically acceptable carriers are chosen based on their biocompatibility, safety, and suitability for the intended route of administration, examples of which include buffers, excipients, fillers, stabilizers, and lubricants.

[0029] The formulation is adjusted to achieve the desired ph, viscosity and osmolarity for the specific route of administration.

[0030] Administration Methods:

[0031] Selected, isolated, purified, or enriched pluripotent migratory MSCs, formulated with a pharmaceutically acceptable carrier, are administered non-invasively along the oro- nasopharyngeal tract by any device or technique capable of depositing a liquid, semi-solid, solid- state, or aerosol dosage form onto nasal, oral, buccal, palatal, pharyngeal, or nasopharyngeal mucosa, thereby placing the cells in intimate contact with richly vascularized surfaces for rapid migration into the systemic circulation.

[0032] Spray, Atomizer, and Inhaler delivery:

[0033] Suitable devices include metered-dose intranasal spray pumps calibrated to discharge about 1 pL to 1 000 pL per actuation; straight or curved (e.g., 45° or 90°) cannula tips for superior-turbinate targeting; and hand-held mucosal atomizers. Pressurized or breath-actuated inhalers— such as propellant-based metered-dose inhalers, soft-mist inhalers, and bidirectional breath-powered nasal devices— may be used alone or with valved holding chambers or spacers.

[0034] Nebulization and Aerosol generation:

[0035] Aerosol delivery may be achieved by vibrating-mesh, ultrasonic, jet, or compressor nebulizers, optionally coupled to nasal or full-face masks, humidifier or vaporizer modules, or pulsating / acoustically enhanced generators that improve paranasal-sinus deposition while maintaining MSC viability in respirable droplets.

[0036] Irrigation, Catheter, and Tube Infusion:

[0037] Large-volume lavage can be provided by squeeze bottles, powered irrigation systems, or flexible nasopharyngeal catheters connected to manual, electronic, or wearable pumps that permit on-demand, continuous, intermittent, or patient-controlled micro-dosing; analogous infusions may be delivered through nasotracheal or endotracheal tubes in conscious, sedated, or ventilated subjects. Direct focal instillation is possible under visual guidance via bronchoscope, laryngoscope, nasal endoscope, or comparable endoscopic instruments is also contemplated.

[0038] Prolonged-Residence Carriers:

[0039] To extend mucosal contact, MSCs may (i) be embedded in pre-formed muco-adhesive hydrogels applied to inferior, middle, or superior turbinates; (ii) be suspended in liquids that gel in situ upon contact with mucosa; or (iii) be incorporated into biodegradable patches, films, microneedle or microneedle-array inserts, nasal stents, tampons, foam plugs, transdermal or sublingual patches, dissolvable palatal / oropharyngeal strips, wafers, lozenges, tablets, pills, or topical creams, ointments, gels, and pastes applied to internal nasal or oropharyngeal surfaces.

[0040] General and Supplementary Applicators:

[0041] Droppers, pipettes, sprayers, insufflators, programmable or patient-controlled micropumps, and any functionally equivalent applicator, irrigator, spray, mist generator, cannula, mask, humidifier, inhaler, nebulizer, atomizer, or transmucosal device known to those skilled in the art are likewise encompassed.

[0042] Embodiments:

[0043] SSEA3+, S1PR2+, CD105+, CD133+MSCs: By enriching the administered MSC population so that >10% co-express SSEA3, S1PR2, CD105 and CD133, the formulation leverages CD133's established role in promoting vascular homing and microvascular engraftment, synergizing with the oro-nasopharyngeal targeted method of delivery, while SSEA3 and S1PR2 synergistically enhance survival and transmigration across the nasal epithelium. CD133 is known to be a marker or enhanced sternness and pluripotency that can enhance the pluripotent, migratory MSC subpopulations ability to differentiate into various cell types in-vivo. This unique four- marker combination is achieved by one or more of ssea3 antibody based selection or cdl33 antibody based selection among umbilical cord tissue derived MSC populations.

[0044] SSEA3+, S1PR2+, CD105+, TERT+MSCs: In a preferred embodiment >10% of administered MSCs additionally express active TERT, such that replicative longevity, telomere maintenance and stress resistance are enhanced, thus enhancing MSC regenerative capacity in vivo. This novel approach, compared to the delivery of conventional MSCs and pluripotent, migratory MSCs lacking telomerase, is enabled by exogenous delivery of modified mRNA encoding the hTERT catalytic subunit, at doses of 0.25-1 pg / mL of mRNA formulated in lipidnanoparticles, thereby avoiding viral integration and preserving genomic integrity, ensuring temporary expression, and mitigating oncogenic risk.

[0045] Non-tumorigenic SSEA3+, S1PR2+, CD105+MSCs with TERT / TERC Inactivation: In a preferred embodiment, to significantly reduce oncogenic risk from the administered cell therapy, a subset of MSCs are rendered permanently telomerase-incompetent via CRISPR-Cas9 knockout of TERT or TERC. These engineered cells still maintain pluripotent, migratory SSEA3 / S1PR2 / CD105 profiles but cannot undergo uncontrolled proliferation, providing the novel safety profile of a "replicative off-switch." This approach is enabled by well-established CRISPR editing protocols and single-cell cloning to confirm bi-allelic inactivation without off-target mutagenesis.

[0046] SSEA3*, S1PR2*, CD105* MSCs Over-expressing HIF-la: In a preferred embodiment, over-expression of HIF-la in pluripotent, migratory MSCs, enabled via significant exposure to hypoxic conditions equal to 3% 02 or lower for greater than or equal to 24 hours primes MSCs for hypoxic survival in-vivo, and increases VEGF secretion and engraftment.

[0047] Neural-Primed MSCs (Nestin*, Tujl* or MAP2+): In a preferred embodiment, >10% of SSEA3+ / S1PR2+ / CD1O5+MSCs are primed or differentiated toward a neural cell phenotype, enabled by BDNF (25-100ng / ml) containing cell culture treatment of MSCs for 3 to 9 days — whereby some MSCs acquire expression of neuronal markers such as Nestin, Tujl or MAP2. This novel neural priming broadens therapeutic applications to specifically enhance neuro- inflammatory or neurodegenerative disorder treatments, and is synergistic with oro- nasopharyngeal administration, particularly with intranasal administration, which has prior shown to be specifically beneficial to target and distribute cell therapies to neural tissue in-vivo, thus allowing for further enhanced neural tissue regeneration and functional engraftment.

[0048] DMSO-Free Cryoprotectant Formulation: In a preferred embodiment the concentrated pluripotent, migratory MSCs are formulated in a defined, sterile, excipient grade, DMSO-free cryoprotectant. Such DMSO-free active cryoprotectant agents may include, but are not limited to, organic osmolytes, organic polymers and albumin. In one embodiment this formulation comprises 5% trehalose, 5% recombinant human albumin and 90% lactated ringers solution —which avoids DMSO-associated toxicity and can serve directly as the final delivery carrier or solution, or as an intermediary for reconstitution in a suitable final formulation. Due to the nontoxic DMSO-free nature of the formulation and ommitance of the requirement for washing away the DMSO prior to administration, less vital cells are lost during such wash steps, less equipment, time and cost is needed at the point of administration, and the viability is increased, such that greater than 95% of post-thawed cells are viable. Such formulations are novel over traditional non-excipient grade or DMSO-based cryopreservation methods and is enabled by pharmaceutically acceptable cryopreservation excipient formulations.

[0049] Supportive Formulation with Senolytic Agent: A preferred embodiment incorporates senolytic(s) (e.g., dasatinib + quercetin) at concentrations previously validated in in-vivo human trials to selectively clear senescent cells, creating a receptive niche for pluripotent, migratory MSC engraftment. The synergistic benefit of combining pluripotent, migratory MSCs with senolysis takes advantage of a unique opportunity wherein the pluripotent, migratory MSCs have also shown to be phagocytic dependent for in-vivo differentiation and dependent on SIP signals for migration and engraftment, which can both be stimulated and provided by the senolytic induced apoptotic cells in-vivo.

[0050] Broad Supportive Formulation: In preferred embodiments the invention provides for co-formulations of MSCs with one or more agents from the classes of absorption enhancers, bronchodilators, mucoadhesives, ECM proteins, osmolytes, cell-death / stress inhibitors, antioxidants, chemokine modulators, anti-inflammatories, biologies, anti-clumping agents, membrane / nucleosome protectants, cytoskeletal remodelers, phagocytosis enhancers, senolytics or oncolytics. This combinatorial approach further enhances the novel cell therapy administration method to further enhance the key attributes of survival, migration, cell health and engraftment which mediate the delivered MSCs benefits.

[0051] Synergistic Dual Agent Formulation: In a preferred embodiment, MSCs are delivered with both an absorption enhancing agent(10 mg / ml hyaluronidase), and a bronchodilating or vasodilating agent(.03% albuterol sulfate), synergistically increasing epithelial penetration, airway patency and vascular migration thereby maximizing the rate of live MSC accumulation into in-vivo tissues in need of healing.

[0052] Oro-nasopharyngeal administration devices and techniques:

[0053] 1. Superior Nasal Turbinate Delivery:

[0054] In a preferred embodiment, the MSC formulation is delivered via an intranasal spray, atomizer, or equivalent device that preferentially directs the formulation specifically to the vascular-rich upper (superior) nasal turbinate region to maximize trans-mucosal uptake into vascular circulation.

[0055] 2. Metered-Dose Intranasal Spray Pump:

[0056] Calibrated bolus delivery (1 - 1000 pL per actuation).

[0057] A commercial positive-displacement pump (e.g., Aptar VP series) can be retro-fitted with a low-shear glass-lined barrel and =0.3 mm spray orifice to generate 30 - 70 pm plumes that preferentially deposit on superior and middle turbinates at a 45° head tilt. The pump reservoir may be siliconized cyclic-olefin polymer (COP) pre-filled with >1 x 107MSCs mL-1in a suitable delivery formulation. One-way valves prevent microbial ingress, enabling multi-dose home use.

[0058] The precise plume geometry maximizes vascular uptake while minimizing lower-airway loss and permits patient-controlled titration (e.g., 5 actuations of 50 pL to reach a 250 pL target dose).

[0059] 3. Pressurized Metered-Dose Inhaler (pMDI) or Soft-Mist Inhaler

[0060] Propellant-free soft-mist cartridges (Boehringer Respimat&type).

[0061] A slow propellant-assisted aerosol of 5 - 30 pm droplets minimizes shear on MSC membranes yet achieves deep penetration into the posterior nasal cavity when fitted with a nasal adaptor. Formulation viscosity is tuned (1.2 - 1.8 cP) with very low molecule weight (<300K Daltons) hyaluronic acid to stabilize droplets and sustain mucosal contact.

[0062] The propellant-free mist reduces barotrauma and allows consistent dose uniformity even at the low actuation forces a frail patient can generate.

[0063] 4. Vibrating-Mesh, Ultrasonic, Jet or Compressor Nebulizers

[0064] Continuous or intermittent aerosolization (0.2 - 1.0 mL min~1).

[0065] Mesh aperture diameters of 5-30 pm yield mass-median aerodynamic diameter (MMAD) particles ideal for nasal-sinus deposition. The device can be coupled to a valved holding chamber that accumulates aerosol during exhalation, thereby conserving expensive cell product.

[0066] This optionally enables programmable micro-dosing via wearable peristaltic pumps that can feed the nebulizer at 1-50 pL min-1, sustaining therapeutic MSC levels over several hours without repeated patient intervention.

[0067] 5. Bi-Directional (Exhalation-Powered) Nasal Delivery

[0068] Devices such as Impel's POD® or Opt-NBridge™.

[0069] During a guided exhalation maneuver the soft palate seals, and aerosol is swept dorsally into the olfactory cleft / superior turbinate. Computational fluid-particle dynamics (CFPD) models show significant increases in the dose reaches the targeted vascular bed versus for straight-through sprays.

[0070] This reduces gastrointestinal drip-loss and exploits highly fenestrated capillaries of the olfactory lamina propria for rapid systemic appearance of MSCs.

[0071] 6. Curved Nasal Spray Cannula (45° or 90° Tip)

[0072] A stainless-steel or medical-grade PEEK cannula with an internal diameter of 0.5 mm and 45° bevel can be attached to a Luer-lock syringe for targeted instillation, with or without endoscopic guidance— useful during outpatient procedures or when precise superiorturbinate placement is desired, required or helpful.

[0073] 7. Acoustic or Pulsating Aerosol Generators

[0074] A device delivering 50-300 Hz pressure oscillations superimposed on the aerosol flow transiently deforms mucus viscoelastic networks, improving paranasal sinus penetration.

[0075] This facilitates delivery to sinonasal reservoirs for improved vasculature uptake and potentially expanding indications (e.g., chronic rhinosinusitis).

[0076] 8. Muco-Adhesive Hydrogels and In-Situ Gelling Liquids

[0077] Pre-formed Hydrogel: A 0.5 % Carbopol® 980 NF solution containing 1 x 107MSCs mL-1is loaded into a aerosolizing device, a unit-dose syringe or gel applicator.

[0078] In-Situ Gel: A 25 % (w / v) Pluronic F127 solution transitions to a gel above 30 °C; when sprayed it forms a 100-300 pm coating that traps MSCs against the epithelium for sufficient time to allow migration into vasculature.

[0079] These extend residence time without significantly impeding viability.

[0080] 9. Biodegradable Patches, Films, Inserts and Microneedle Arrays

[0081] Chitosan-azelate (Chi-Aze) Patch: A 100 pm porous film can uptake roughly hold up to 5 x 106MSCs cm-2.

[0082] Microneedle Array: Poly-l-lactic acid (PLLA) or maltose needles 400 pm tall (density 100 needles cm-2) breach the superficial epithelium, depositing MSCs directly into lamina propria blood vessels.

[0083] These provides zero-order (diffusion-controlled) cell release for extended time periods, protect the cell formulation from losing proximal access to mucosal surface and vasculature, and enhance vasculature access.

[0084] 10. Palatal & Buccal Dissolvable Strips, Wafers or Lozenges

[0085] Fast-dissolving pullulan films (50 pm) incorporating refined trehalose:mannitol (1:1) can preserve MSC membrane integrity during drying. Upon oral, buccal or palatal administration salivary dissolution releases cells that migrate through microvasculature.

[0086] 11. Catheter-Based and Endoscopic Instillation

[0087] A flexible nasopharyngeal catheter coupled to a syringe pump can support continuous micro-infusion (5-120 pL min-1) in ventilated or conscious patients. Real-time endoscopy ensures accurate placement.

[0088] 12. Nasal Stents, Tampons, Foam Plugs

[0089] A Collagen or Gelatin foam, pre-soaked or coated with MSC formulation and expanded in situ to provide MSCs contact and access to internal nasal mucosa, can slowly release the cell payload over 30 minutes to 8 hours, thereafter being removable.

[0090] 13. Humidifiers or Vaporizers

[0091] A low-temperature (<40 °C) bubble humidifier fitted with sterile-air filters can mix aerosolized MSCs into inhaled humidified gas streams for patients, fitted with nasal or face masks for inhalation.

[0092] 14. Topical Nasal Creams, Ointments, Gels or Pastes

[0093] A water-in-oil cream containing 5 % phosphatidylcholine liposomes, or a 0.1% Very Low Molecular Weight (<300K Daltons) Hyaluronic Acid Hydrogel formulation enhances MSC retention and mucosal adhesion and specific locations of oro-nasopharyngeal placement with an easy and controlled viscous applicator.

[0094] 15. Programmable Wearable Pump Delivery

[0095] In some preferred embodiment MSCs are delivered via a manual, automated or programmable dispenser tethered to a wearable pump, the system enables on-demand microdosing— continuous, intermittent or patient-controlled— thereby precisely matching cell delivery kinetics to fluctuating physiologic needs (e.g., post-injury inflammatory peaks), which is novel over single bolus administration and fully enabled by adapting established insulin-pump and wearable-infusion technologies (e.g., peristaltic or piston-driven microfluidic pumps) with gentle, low-shear flow.

[0096] Dose Regimen and Self-Administration:

[0097] In a preferred embodiment, the dose regiment prescribes the administration of >1 x 106pluripotent, migratory MSCs within a 24 hour time window. In another preferred embodiment the delivery method and device is formulated for safe and convenient self-administration via a patient-friendly nasal spray, eliminating the need for a healthcare professional or clinical setting.

[0098] For intranasal self-administration the patient would tilt their head 30-45° backward, exhale, actuate during a 2s breath-hold, then maintain soft-palate closure for 10 s.

[0099] The self-administrable delivery device and open-access instructions ensure that this method is fully enabled for "at-home" and outpatient use.

[0100] Variable and Controlled Dosing Regimens: By concentrating pluripotent, migratoryMSCs and then administering them according to rapid or programmable regimens— continuous,gradual, variable or intermittent dosing over hours to days— this approach affords control over in vivo cell kinetics to optimize engraftment and paracrine activity based on the patients own unique and adapting response over time to the live cell therapy, and takes advantage of the painless, non-invasive, simple, low cost, and at-home delivery methods detailed herein.

[0101] Administration Regimens:

[0102] Dosage regimens may range from single administrations to continuous or intermittent delivery over extended periods (seconds, minutes, hours, days, weeks, months, or years), tailored to therapeutic objectives or prophylactic needs. Due to the non-invasive, simple, low cost and low resource requirements of such delivery methods, the intended recipient may freely administer the cell therapy to themselves, with or without the aid of a medical professional, as often as needed or desired.

[0103] In order to achieve a therapeutically beneficial effect, the preferred dose range prescribed for this method is at least 1 million pluripotent, migratory stem cells administered within a 24 hour period. In a preferred embodiment, for liquid or gel suspension based formulations and delivery, 1 million pluripotent, migratory stem cells would be suspended per 100 microliters of a pharmaceutically acceptable formulation as described.

[0104] Summary of Benefits:

[0105] Bypass Pulmonary First-Pass: IV administered MSCs are largely trapped in lung capillaries; oro-nasopharyngeal administration reduces lung sequestration. Pluripotent, migratory MSCs are uniquely suited for a high rate of migration into vascular circulation when administered to the oro-nasopharyngeal tract. Blood Brain Barrier effects are also reduced via chosen methods of administration, such as to the upper-nasal vascular bed, when so desired to increase the rate of accumulation in brain tissue and brain vasculature.

[0106] Upper-Nasal Vascular Bed: Highly fenestrated veins (Kiesselbach plexus) enable rapid transmigration in to vascular circulation.

[0107] Immune-Quiescent Interface: Nasal mucosa expresses tolerogenic Dendritic Cells, which supports survival of allogeneic MSCs.

[0108] Patient convenience and efficacy: Self-administration possible, non-invasive, minimal resources and technical know-how, short duration administration possible, on-demand dosing enabled, prophylactic dosing enabled, and generally improves dosing frequency potential and total cells administered over longer periods of time for chronic indications.

[0109] Clinical Applications:

[0110] This invention is advantageous for the general health of the treated person, including but not limited to general health maintenance, prophylaxis against degenerative, treatment of inflammatory or infectious conditions, treatment of acute or chronic inflammation, active tissue repair following injury or degenerative damage, tissue repair for chronic degenerative disorders, ameliorating genetic disorders, pain management, and treatment for autoimmune disorders, neurological disorders, infectious diseases, organ failure, frailty, wound healing and cosmetic applications.

Claims

Claims1. A method for increasing the number of live, migratory mesenchymal stem cells (MSCs) in circulation in a human being, comprising: a. Selecting, isolating, purifying or enriching a specific pluripotent, migratory MSC subpopulation characterized by surface markers SSEA3+, S1PR2+, and CD105+; b. Formulating the selected, isolated, purified or enriched cell population comprising at least 10% concentration of pluripotent, migratory MSCs among the total MSCs into a suitable formulation comprising cryoprotectants, delivery or carrier solutions; c. Loading the concentrated pluripotent, migratory MSC formulation into a suitable delivery device; d. Administering sufficient quantity of said selected, isolated, purified or enriched cell population via Oro-Nasopharyngeal delivery to a human being via a suitable delivery technique, thereby effectively increasing the number of live, migratory mesenchymal stem cells (MSCs) in the treated person's circulation.

2. The method of claim 1, wherein the administered MSC population comprises at least about 20% SSEA3+, S1PR2+, CD105+ MSCs.

3. The method of claim 1, wherein the administered MSC population comprises at least about 50% SSEA3+, S1PR2+, CD105+ MSCs.

4. The method of claim 1, wherein the administered MSC population comprises at least about 75% SSEA3+, S1PR2+, CD105+ MSCs.

5. The method of claim 1, wherein the administered MSC population comprises at least about 90% SSEA3+, S1PR2+, CD105+ pluripotent, migratory MSCs.

6. The method of claim 1, wherein at least 10% of the administered MSC population comprises non-immunogenic SSEA3+, S1PR2+, CD105+, HLA-G+, HLA-B negative MSCs.

7. The method of claim 1, wherein at least 10% of the administered MSC population comprises SSEA3+, S1PR2+, CD105+, CD133+ MSCs.

8. The method of claim 1, wherein at least 10% of the administered MSC population comprises SSEA3+, S1PR2+, CD105+, TERT+ MSCs.

9. The method of claim 1, wherein at least 10% of the administered MSC population comprises non-tumorigenic SSEA3+, S1PR2+, CD105+, MSCs which have been genetically engineered with a knockout or inactivation of the TERT or TERC gene.

10. The method of claim 1, wherein at least 10% of the administered MSC population comprises SSEA3+, S1PR2+, CD105+ MSCs which also over-express HIF-la at levels significantly higher than bulk MSCs cultured in normoxic environments.

11. The method of claim 1, wherein at least 10% of the SSEA3+, S1PR2+, CD105+ MSCs of the administered MSC population also express at least one of the neural stem cell marker selected from Nestin+, Tujl+ or MAP2+.

12. The method of any preceding claim, wherein the concentrated pluripotent, migratory MSCs are formulated in a DMSO-free, sterile, excipient grade, cryoprotectant formula, which is either: A) itself suitable as a final pharmaceutical formulation solution and carrier for administration; or B) used as an intermediary formulation, which is to be thawed after cryopreservation and then reconstituted or replaced in a delivery or carrier solution prior to delivery device loading and subsequent administration.

13. The method of any preceding claim, wherein the administered formulation in a preferred embodiment comprises a supportive formulation comprising at least one senolytic agent.

14. The method of any preceding claim, wherein the administered formulation comprises a supportive formulation comprising one or more agents selected from absorption-enhancing agents, bronchodilating agents, mucoadhesive polymers, extracellular matrix proteins, osmolytes, cell death inhibitors, cell stress inhibitors, antioxidants, chemokine receptor modulators, anti-inflammatory agents, biologic agents, anti-clumping agents, membrane protective or repairing agents, nucleosome protective or repairing agents, cytoskeletal rearrangement enhancing agents, phagocytosis enhancing agents, senolytic agents or oncolytic agents.

15. The method of any preceding claim, wherein the administered formulation in a preferred embodiment comprises a supportive formulation simultaneously and synergistically comprising an absorption enhancing agent and a bronchodilating or vasodilating agent.

16. The method of any preceding claim, wherein the administered formulation in a preferred embodiment comprises a supportive formulation comprising a senolytic agent.

17. The method of any preceding claim, wherein the number of administered pluripotent, migratory MSCs is at least 1,000,000 in a 24 hour window.

18. The method of any preceding claim, wherein the MSCs are self-administered without a medical professional.

9. The method of any preceding claim, wherein the oro-nasopharyngeal administration of theMSCs is effected using one or more of the following devices or techniques: a. a metered-dose intranasal spray pump calibrated to deliver 1 - 1000 piL per actuation of the MSC formulation; b. a pressurized metered-dose inhaler (pMDI) or soft-mist inhaler, optionally used with a valved holding chamber or spacer, delivering the MSC formulation to the nasal cavity; c. a vibrating-mesh, ultrasonic, jet or compressor aerosol generator or nebulizer coupled to a nasal mask or face mask and generating aerosol particles to deliver the MSC formulation; d. an exhalation- or breath-powered bi-directional nasal delivery device that directs aerosolized MSCs to the nasal or upper nasal cavity; e. an atomization device, or mucosal atomization device, fitted to a suitable container or syringe containing the MSC formulation, that directs aerosolized MSCs to the nasal or upper nasal cavity; f. A curved nasal spray cannula (e.g., 45° or 90° tip) for precise superior-turbinate targeting; g. a pulsating or acoustically-enhanced aerosol generator for improved paranasal sinus penetration; h. a pre-formed muco-adhesive hydrogel containing the MSCs that is swabbed, sprayed or dispensed onto the inferior, middle or superior nasal turbinate region; i. an in-situ gelling liquid containing the MSCs that solidifies upon contact with oro- nasopharyngeal mucosa to prolong residence of the MSCs; j. a biodegradable patch, film or insert (including micro-needle, microneedle-array or hydrogel-loaded formats) placed in contact with the oro-nasopharyngeal mucosa, including but not limited to being placed intranasal, sublingual, buccal, on the palate, or trans-oral; k. a multi-purpose spray nozzle, dropper or pipette delivering the MSC formulation to the oro-nasopharyngeal tract l. a palatal or oropharyngeal dissolvable strip, wafer or lozenge that releases MSCs across mucosa m. a nasal irrigation lavage bottle or system dispensing the MSC formulation;n. a flexible nasopharyngeal catheter connected to a manual or electronic pump for administration of the MSC formulation; o. a bronchoscope, laryngoscope, nasal endoscope or similar endoscopic device for direct instillation of MSCs onto oro-nasopharyngeal mucosa; p. a nasotracheal tube delivering the MSC formulation to conscious, sedated or ventilated subjects; q. a nasal stent, tampon or foam plug impregnated or coated with the MSC formulation; r. an aerosol inhaler attached to a breath-actuated nasal or inhaler containing the MSC formulation; s. a humidifier or vaporizer coupled to a nasal mask or face mask and generating aerosol particles to deliver the MSC formulation; t. a topical nasal cream, ointment, gel or paste containing the MSC formulation applied to internal nasal or oropharyngeal surfaces; u. any functionally equivalent nasal, oral, buccal, or pharyngeal applicator, irrigator, spray, swab, mist-generator, cannula, mask, humidifier, inhaler, nebulizer, atomizer, insufflator or transmucosal dosage delivery devices or techniques known in the art.

20. The method of any preceding claim, wherein MSC delivery is achieved via a manual, automated or programmable dispenser connected to manual or electronic wearable pump, enabling on- demand, continuous, intermittent or patient-controlled micro-dosing of the MSC formulation via any of the aforementioned routes, techniques or devices.

21. The method of any preceding claim, wherein the pluripotent, migratory MSCs are concentrated by one or more method of selective stress tolerance resistance based selection or antibody based selection, and thereafter administered via a rapid method or via a continuous, gradual, variable, and / or intermittent method, administered once or many times, such as to achieve a desired or optimal therapeutic outcome by controlling dosing over any length of period of time, via methods known to one skilled in the art.

22. The use of the method of any preceding claim for enhancing the health or appearance of the treated person, including but not limited to general health maintenance, prophylaxis against degenerative, inflammatory or infectious conditions, treatment of acute or chronic inflammation, active tissue repair following injury or degenerative damage, tissue repair for chronic degenerative disorders, ameliorating genetic disorders, pain management, autoimmunedisorders, neurological disorders, infectious diseases, organ failure, frailty, wound healing, and cosmetic applications.