The science behind our services
Mesenchymal Stromal Cells (MSCs).
An overview of the cell biology underlying autologous bone-marrow–derived therapies, definitions, cell-size and the first-pass effect, allogeneic immunogenicity, extracellular vesicles, senescence, and the blood–brain barrier.
01 · Definition
MSC Classification
The International Society for Cellular Therapy (ISCT) Mesenchymal and Tissue Stem Cell Committee defines mesenchymal stem cells by three minimal criteria. A cell population must meet all three to be classified as MSCs:
- Plastic adherence. The cells must adhere to plastic under standard culture conditions.
- Tri-lineage differentiation. When stimulated in the laboratory, the cells must be able to differentiate into bone (osteoblasts), fat (adipocytes), and cartilage (chondroblasts).
- Surface-marker expression. The cells must express a specific surface-marker phenotype, positive for CD105, CD73, and CD90; negative for CD45, CD34, CD14, CD19, and HLA-DR (detailed below).
MSCs are further classified by cell source, bone marrow–derived (BM-MSCs), adipose-derived (AD-MSCs), and umbilical cord MSCs (UC-MSCs). Regardless of source, CD105, CD73, and CD90 are expressed on virtually all mesenchymal stem cells. MSCs are multipotent spindle-shaped cells with self-renewal capacities that can differentiate into related cell types in vitro or by transplantation.1
More recently, it has been found that MSCs play a more significant role in producing regenerative and immunomodulatory elements under normal conditions, as opposed to differentiation in vivo.2
In plain language
The clinical signal from MSCs comes from the messages they send, paracrine signaling, immune modulation, support of local repair, not from MSCs turning into new cartilage, neurons, or heart cells in your body. This matters scientifically and shapes how the FDA categorizes the therapy.
Figure 1 · Spindle shaped, plastic-adherent MSCs in a cultured laboratory environment.
02 · Cell size
The first-pass effect.
An often-overlooked issue in clinical intravenous human MSC (hMSC) treatments revolves around the diameter and volume of the cells being infused. The first-pass effect is a known phenomenon in the lungs3, mechanical trapping of infused cells in the pulmonary capillaries.
The first-pass effect occurs if the infused MSCs are larger in diameter than the pulmonary capillaries (10–15 µm). Ex-vivo culture and expansion of BM-hMSCs leads to increases in cell diameters and volumes, surpassing the pulmonary capillaries' diameters. Bhat et al.4 reported that the average diameter of low-serum cultured BM-hMSCs is between 16.02 (±0.16) and 19.2 (±0.22) µm.
Evidence suggests that in humans, the entrapped cells in the lungs are subsequently cleared and have been found to accumulate in the spleen and liver over the ensuing hours to days after infusion.5 The fate of the cleared and redistributed cells is concerning: it is now thought to be due to the phagocytic work of monocytes, resulting in apoptosis of the infused MSCs.6
Endogenous MSCs in the bone marrow are approximately 10 µm in size, enabling an enhanced ability to navigate and pass through capillaries, particularly the lungs. In contrast, undifferentiated adipose-derived stem cells (AD-SCs) are larger (mean diameter ~14.9 µm), which effectively puts AD-SCs at the upper limit of the pulmonary capillary bed diameter.
| Culture state | Mean diameter | Relative to pulmonary capillary (10–15 µm) |
|---|---|---|
| Endogenous BM-MSC (reference) | ~10 µm | Below, navigates capillary bed |
| Undifferentiated AD-MSC | ~14.9 µm | At upper end of capillary bed |
| Differentiated / activated AD-MSC | ↑ with culture days | Above, first-pass trapping likely |
| Low-serum cultured BM-hMSC (Bhat 2021) | 16.0–19.2 µm | Above, first-pass trapping likely |
Clinical implication
From the standpoint of pulmonary capillary diameters and trapping-associated first-pass effect, undifferentiated BM-MSCs are the logical choice for IV-infusion–based treatments. Therapeutic AD-MSCs and differentiated BM-MSCs are best reserved for a localized, targeted approach to the desired tissues.
03 · Immunogenicity
Allogeneic vs autologous, revisiting "immune-privileged."
In the past, allogeneic MSCs were thought to be immune-privileged due to their lack of major histocompatibility complex (MHC) class II molecules. Results from more recent studies have shown that in vivo administration of allogeneic MSCs may provoke a strong immune response in the host.8
Although culture-expanded MSCs express low levels of MHC class I and no MHC class II, when MSCs differentiate into mature cells or are exposed to IFN-γ, they express much more MHC class I and start to express MHC class II.9 These conditions correlate with what occurs in the in vivo setting after MSC administration. The growing body of evidence suggests that increased allogeneic MSC immunogenicity occurs with induced differentiation: which necessitates revisiting the held notion that MSCs are immune-privileged. These issues do not concern induced differentiation of autologous MSCs, since any MHC expression is native to the host recipient.
Joswig et al.10 evaluated the response to repeated intra-articular injections of autologous versus allogeneic BM-MSCs in an equine model. Following the second dose, the allogeneic BM-MSC recipients had elevated total nucleated cell counts in the injected joints, while no adverse immunogenic responses were seen in the autologous group. This should prompt further consideration about how to best proceed in patients requiring multiple administrations of allogeneic stem cell–based therapies.
04 · Signaling cargo
Extracellular vesicles & exosomes.
All living cells release extracellular vesicles (EVs) derived from bilayer lipid membranes. EVs transport many cell-derived molecules, nucleic acids, proteins, lipids, and metabolites, that reflect the originating cell's characteristics. Subtypes include exosomes, ectosomes, oncosomes, microvesicles, and apoptotic bodies. L. Ramos et al.11 characterized EVs released from BM-hMSCs as positive for markers CD44, CD73, and CD90.
Exosomes are mediators of intercellular communication by microRNA, the smallest EVs, with a diameter range of 30–150 nm. Exosomes are formed by exocytosis of multivesicular bodies (MVBs), which frees intraluminal vesicles by fusing with the plasma membrane.13 The exosomal membrane is characterized by sphingomyelin, cholesterol, ceramide, and phosphatidylserine.
Recent research has revealed that exosomes may transfer bioactive molecules, pointing to a prominent role in the pathologies of Parkinson's,14 Alzheimer's,15 spongiform encephalopathies,16 and ALS.17 This same bioactive-transport capacity may also be therapeutic: MSC-derived exosomes can home to pathologic areas of the brain to reduce levels of inflammation and Alzheimer's-specific amyloid-β plaques.18
Why this matters clinically
The small size of exosomes and EVs (down to ~30 nm) facilitates passage through the smallest capillaries (~1 µm): they reach areas in the body cells cannot. That's the therapeutic upside: a microscale, cell-derived delivery system for bioactive signals.
05 · Senescence
Age, disease & culture-related stem cell impairment.
Throughout an individual's lifespan, stem cells maintain a continued presence in the body, making them prone to cell damage, loss of regenerative function, senescence (inability to proliferate), and cell death.19 The buildup of toxic metabolites is theorized to be central to accumulative DNA and macromolecule damage.20 Aging is also implicated in alterations in protein homeostasis21 and mitochondrial DNA mutations22 that deplete the stem-cell pool.
Aging-related decline affects MSCs, hematopoietic stem cells (HSCs), neural stem cells (NSCs), skeletal muscle progenitor cells (SMPCs), skin stem cells (SSCs), and germ-line stem cells.23 Specific to MSCs, age-dependent bone marrow declines have been found alongside functional alterations: impairments in migratory capacity and senescence tendencies.24
Age-related stem cell impairment parallels what is found with chronic diseases:
- Diabetes
- Obesity
- Dyslipidemia
- Glucocorticoid excess
- Rheumatoid arthritis (RA)
- Systemic lupus erythematosus (SLE)
- Cardiovascular disease (CVD)
- Osteoporosis
Specific stem-cell lines impacted by each are detailed in Table 2 of Pérez, de Lucas, and Gálvez.25
Hayflick limit and culture-induced aging.
To augment clinical utility, MSCs are frequently expanded in vitro to achieve appropriate cell numbers. Population doublings (PDs) serve as a quantitative reference for culture expansion. Despite the source, MSCs demonstrate replicative senescence in vitro after repeated PDs, the Hayflick limit.46 The estimated maximum possible PDs before replicative cessation is 30–40, and even minimal passaging induces rapid MSC aging through telomere shortening.47
Expansion-induced aging raises the question of whether therapeutic emphasis should be placed on quantity or quality of MSCs employed in treatments. In a meta-analysis by Muthu et al.48 of 17 RCTs studying autologous MSC treatment in knee OA (767 patients, both cultured and uncultured):
| Cohort | Time point | Cultured expanded (CE) | Uncultured (UC) |
|---|---|---|---|
| VAS | 6 months | Significant | Not significant |
| VAS | 12 months | Not significant | Significant |
| WOMAC | 12 months | Not significant | Significant |
| KOOS | 12 months | Not significant | Significant |
Takeaway
By the p<0.05 standard, uncultured MSCs outperformed both controls and culture-expanded cells at 12 months on VAS, WOMAC, and KOOS scores. Heterogeneity in pooled studies limits the meta-analysis, but the direction of evidence supports quality over quantity for autologous knee-OA work.
Heterogeneity, CFUs & senescence markers.
MSC heterogeneity encompasses proliferation rate, immunophenotype, morphology, multilineage differential potential, and senescence.49 The number of colony-forming units (CFUs) consistently decreases with culture expansion, and CFU numbers become negligible after 20 passages.51
Morphologically, large MSCs in culture with increased granularity coincide with senescence.52 MSC autofluorescence has been put forth as a real-time, non-invasive, and quantifiable senescence marker.53 Other markers include β-galactosidase54 and senescence-associated α-L-fucosidase. Since telomere length decreases with each cell division until a critical-length senescence stage, telomeres predict senescence and estimate division history.55
The current focus is on approaches that undo or mitigate senescence, induced pluripotent stem cell–derived MSCs (IMSCs), genetic engineering targeting senescence-involved molecules, and pharmacologic approaches like rapamycin, which inhibits mTOR.
06 · CNS access
The blood–brain barrier.
The blood–brain barrier (BBB) is the network of endothelial cells forming the central nervous system's highly selective barrier. It generally facilitates molecular transport into the brain by lipid-solubility diffusion and catalyzed transport. The BBB plays an essential role in normal brain function by maintaining homeostasis and immune-cell transport, and protects the CNS from toxins, pathogens, injury, inflammation, and disease.56 Any compromise in BBB selectiveness may allow pathogens into the CNS and cause neuroinflammation, contributing to neurodegenerative-disease progression.57
By default, a limited number of solutes can passively cross the BBB without active transport, gases (O₂, CO₂), low-molecular-weight lipid-soluble molecules under 400 Da, or molecules with fewer than eight hydrogen bonds.58
The degree to which the BBB represents an active barrier to MSCs entering the CNS has not been fully elucidated. Yet recent research has revealed that MSCs can cross endothelial cells using transcellular or paracellular transport to exert therapeutic benefits.59
MSC homing may occur as a systemic or nonsystemic process. Nonsystemic homing represents direct inoculation into targeted tissues, where local cytokines guide cells to injured areas. Systemic homing refers to administration away from injured tissues.60 Several models describe the mechanism, MSCs leaving the circulation by endothelial integration, migration through the endothelial barrier, basement-membrane penetration, and formation of plasmic podia to infiltrate target tissues.61
Why this informs our clinical framing
Intranasal delivery, via olfactory and trigeminal pathways, is being investigated specifically because it bypasses systemic circulation and the BBB as a gating constraint. It remains investigational, not FDA-approved for neurological disease, and is discussed as a potential access route, not a proven therapeutic pathway.
References
Selected citations.
A working bibliography drawn from the live article. Full citation list available on the live patient site.
- Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. ISCT position statement. Cytotherapy. 2006;8:315–7.
- Guimarães-Camboa N, Cattaneo P, Sun Y, et al. Pericytes of multiple organs do not behave as mesenchymal stem cells in vivo. Cell Stem Cell. 2017;20:345–359.e5.
- Toma C, Wagner WR, Bowry S, et al. Fate of culture-expanded MSCs in the microvasculature. Circ Res. 2009;104:398–402.
- Bhat S, Viswanathan P, Chandanala S, et al. Expansion and characterization of BM-MSCs in serum-free conditions. Sci Rep. 2021;11:3403.
- Krueger TE, Thorek DL, Denmeade SR, et al. MSC-based drug delivery: the good, the bad, the ugly, and the promise. Stem Cells Transl Med. 2018;7(9):651–663.
- Galleu A, Riffo-Vasquez Y, Trento C, et al. Apoptosis in MSCs induces in vivo recipient-mediated immunomodulation. Sci Transl Med. 2017;9:eaarn7828.
- MilliporeSigma. Rapid Analysis of Human Adipose-Derived Stem Cells and 3T3-L1 Differentiation.
- Ankrum JA, Ong JF, Karp JM. MSCs: immune evasive, not immune privileged. Nat Biotechnol. 2014;32(3):252–60.
- Joswig AJ, Mitchell A, Cummings KJ, et al. Repeated intra-articular allogeneic MSC injection causes adverse response vs autologous in equine model. Stem Cell Res Ther. 2017;8(1):42.
- Joswig AJ, et al. (companion citation, same study).
- L Ramos T, Sánchez-Abarca LI, Muntión S, et al. MSC surface markers identify MSC-derived EVs by flow cytometry. Cell Commun Signal. 2016;14:2.
- Sidhom K, Obi PO, Saleem A. Exosomal isolation methods. Int J Mol Sci. 2020;21(18):6466.
- Kowal J, Tkach M, Théry C. Biogenesis and secretion of exosomes. Curr Opin Cell Biol. 2014;29:116–125.
- Emmanouilidou E, et al. Cell-produced α-synuclein secreted via exosomes. J Neurosci. 2010;30:6838–6851.
- Rajendran L, et al. Alzheimer's β-amyloid peptides released via exosomes. PNAS. 2006;103:11172–11177.
- Fevrier B, et al. Cells release prions in association with exosomes. PNAS. 2004;101:9683–9688.
- Rajendran L, et al. (companion citation).
- Perets N, Betzer O, Shapira R, et al. Golden exosomes selectively target brain pathologies. Nano Lett. 2019;19(6):3422–3431.
- Yun MH. Changes in regenerative capacity through lifespan. Int J Mol Sci. 2015;16:25392–25432.
- Yang SR, Park JR, Kang KS. Reactive oxygen species in MSC aging. Oxid Med Cell Longev. 2015;2015:486263.
- Tomaru U, et al. Decreased proteasomal activity and age-related phenotypes. Am J Pathol. 2012;180:963–972.
- Bratic A, Larsson NG. The role of mitochondria in aging. J Clin Invest. 2013;123:951–957.
- Schultz MB, Sinclair DA. When stem cells grow old. Development. 2016;143:3–14.
- Kasper G, et al. Insights into MSC aging. Stem Cells. 2009;27(6):1288–97.
- Pérez LM, de Lucas B, Gálvez BG. Unhealthy stem cells: when health conditions upset stem-cell properties. Cell Physiol Biochem. 2018;46(5):1999–2016.
- Hayflick L, Moorhead PS. Serial cultivation of human diploid cell strains. Exp Cell Res. 1961;25:585–621.
- Baxter MA, et al. Telomere length and rapid aging of human marrow stromal cells. Stem Cells. 2004;22:675–682.
- Muthu S, et al. Is culture expansion necessary in autologous MSC therapy for knee OA?, meta-analysis. Bioengineering. 2021;8(12):220.
- Schellenberg A, et al. Population dynamics of MSCs during culture expansion. Cytotherapy. 2012;14:401–411.
- Block TJ, et al. Restoring quantity and quality of elderly human MSCs. Stem Cell Res Ther. 2017;8:239.
- Bertolo A, et al. Autofluorescence as a reliable in-vitro marker of MSC senescence. Sci Rep. 2019;9:2074.
- Dimri GP, et al. A biomarker that identifies senescent human cells. PNAS. 1995;92:9363–9367.
- Daneman R, Prat A. The blood–brain barrier. Cold Spring Harb Perspect Biol. 2015;7:a020412.
- Graham NSN, Sharp DJ. Understanding neurodegeneration after TBI. J Neurol Neurosurg Psychiatry. 2019;90(11):1221–1233.
- Pardridge WM. Drug transport across the blood–brain barrier. J Cereb Blood Flow Metab. 2012;32:1959–72.
- Matsushita T, et al. MSCs transmigrate across brain microvascular endothelial monolayers. Neurosci Lett. 2011;502(1):41–45.
- Chia YC, et al. Stem cell therapy for neurodegenerative diseases, bypassing the BBB. Stem Cells Int. 2020:8889061.
- Steingen C, et al. Key mechanisms in MSC transmigration and invasion. J Mol Cell Cardiol. 2008;44(6):1072–1084.
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