Cellular Therapies - Science-to-Therapeutic Application

Jason Glowney, MD, MSc

Mesenchymal Stem Cells (MSCs)

Mesenchymal stem cells (MSCs) are classified by cell source, which includes bone marrow-derived (BM-MSCs), adipose-derived (ASCs), and umbilical cord MSCs. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy[1] defines mesenchymal stem cells as positive for CD105 (ENG), CD73 (NT5E), and CD90 (THY1), and negative for CD45, CD34, CD14, CD19, and HLA-DR. 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. 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].

An often-overlooked issue in clinical intravenous human mesenchymal stem cell (hMSC) treatments revolves around the diameter and volume of the MSCs being infused into the patient. The 'first-pass effect' is a known phenomenon in the lungs[3], representing the 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 10-15um. The ex vivo culture and expansion of BM-hMSCs lead to increases in cell diameters and volumes, surpassing the pulmonary capillaries' diameters. Bhat[4] et al. reported that the average diameter of low serum cultured BM-hMSCs is between 16.02 (+/- 0.16) and 19.2 (+/-0.22)um. 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, and it is now thought to be due to the phagocytic work of monocytes, resulting in the apoptosis of the infused MSCs[6].

Endogenous MSCs in the bone marrow are approximately 10um in size, enabling an enhanced ability to navigate and pass through capillaries in the body, particularly the lungs. In contrast, undifferentiated adipose-derived stem cells (AD-SCs) are larger and have a reported mean diameter of approximately 14.9um, which effectively puts AD-SCs at the upper limit in the range of the pulmonary capillary bed diameter. Like BM-MSCs, activated ADSCs have greater mean cell diameters than undifferentiated ADSCs, where increases in diameter are positively correlated with the number of differentiation days:

From the standpoint of pulmonary capillary diameters and the cell trapping-associated 'first-pass effect,' undifferentiated BM-MSCs are, in theory, the logical choice for IV-infusion-based stem cell treatments in patients. The application of therapeutic undifferentiated/differentiated ADSCs and differentiated BM-MSCs is best reserved for a localized and targeted approach to treating the desired tissues.

In the past, allogenic 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 allogenic 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-, they express much more MHC class I and will start to express MHC class II[9]. These conditions correlate with what occurs in the in vivo setting for the MSCs after their administration to the recipient. So, 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 the induced differentiation of autologous MSCs, given that any MHC expression would be native to the host recipient.

Joswig et al.[10] evaluated the response to repeated intra-articular injections of autologous versus allogeneic BM-MSCs using an equine model. Following the second dose, the allogeneic BM-MSCs 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 in their treatment course.

Extracellular Vesicles (EVs) and Exosomes

All living cells release extracellular vesicles (EVs) derived from bilayer lipid membranes. EVs transport many cell-derived molecules, including nucleic acids, proteins, lipids, and metabolites that reflect the originating cell's characteristics. Extracellular vesicles refer to secreted cellular vesicles in general, and several EV subtypes have been identified and defined, including exosomes, ectosomes, oncosomes, microvesicles, and apoptotic bodies. L. Ramos et al.[11] characterized extracellular vesicles (EVs) released from BM-hMSCs as less than and positive for markers CD44, CD73, and CD90.

Exosomes are mediators of intercellular communication by microRNA and are the smallest EVs, having a diameter range of 30-150nm. Exosomes are formed by the 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, which can be employed in their identification process. Recent research has revealed that exosomes may also transfer bioactive molecules, which leads to their potential to play a prominent role in the pathologies of Parkinson's Disease[14], Alzheimer's[15], spongiform encephalopathies[16], and ALS[17]. The capacity of exosomes to transport bioactive molecules may also be therapeutic, where MSC-derived exosomes can home to pathologic areas of the brain to reduced levels of inflammation and Alzheimer’s specific amyloid beta plaques[18]. The upside potential for exosome treatments is to use this bioactive molecule transport capacity as a microscale therapeutic agent delivery system. The small size of exosomes, and EVs in general, facilitates their passage through the smallest-sized capillaries in humans (approx.=1um), underscoring their therapeutic potential to reach all areas in the body.

Age, Disease, and Culture-Related Stem Cell Impairment and Senescence

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 homeostasis[21] and mitochondrial DNA mutations[22] that deplete the stem cell pool and negatively impact their function. To date, aging-related decline and impairment are known to affect 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, along with functional alterations that include impairments in migratory capacity and senescence tendencies[24].

Age-related stem cell impairment parallels that which is found with chronic diseases, including:

• Diabetes

• Obesity

• Dyslipidemia

• Glucocorticoid Excess

• Rheumatoid Arthritis (RA)

• Systemic Lupus Erythematosus (SLE)

• Cardiovascular Disease (CVD)

• Osteoporosis

The specific stem cell lines impacted by each chronic disease listed above are shown in the table below and adapted from the work of Pérez, de Lucas, and Gálvez[25].

 To augment their clinical utility, MSCs are frequently expanded in vitro to achieve appropriate cell numbers for therapeutic treatments. Population doublings (PDs) serve as a quantitative reference for the culture expansion of cells, and in the case of MSC expansion, this typically involves numerous passages. Despite the source, MSCs will demonstrate replicative senescence in vitro after repeated PDs, known as the Hayflick limit[46]. The estimated maximum possible number of PDs before replicative cessation occurs for MSCs in vitro is 30-to-40, and even minimal passaging induces rapid MSC aging through telomere shortening[47].

Expansion-induced MSC aging raises the question of whether therapeutic emphasis should be placed on the quantity or quality of the MSCs employed in treatments. In a meta-analysis by Muthu et al.[48], 17 RCT studies were included that studied the clinical effectiveness (VAS/WOMAC/KOOS/Lysholm scores) of autologous MSC treatment in humans with knee osteoarthritis (767 total patients) and included adipose-derived and bone marrow-derived MSCs, both cultured and uncultured. The efficacy outcomes included 6 and 12-month patient follow-ups for the included studies using VAS and WOMAC assessment scale cohorts and a 12-month patient follow-up KOOS and Lysholm scale cohorts, which are presented in the following table:

 By the oft-used standard for statistically significant probability of p < 0.05, only 4 subgroups are statistically significant in the three cohorts, those being for the cultured expanded cells at 6 months and the uncultured cells at 12 months in the VAS cohort, the uncultured cells at 12 months in the WOMAC cohort, and the uncultured cells at 12 months in the KOOS cohort. By the standard p < 0.05, we can surmise that uncultured stem cells were more effective than study controls at 12 months by VAS, WOMAC, and KOOS scores. UCs also showed more significant improvements in WMD scores than CEs using VAS, WOMAC, and KOOS scales. However, this must be interpreted considering the CE cohorts did not achieve statistically significant probabilities of p <0.05 for any cohort at 12 months. At 6 months, UCs had better WMDs than study controls and CEs for the VAS and WOMAC cohorts, except for the 6 months VAS CE group, all p-values did not achieve the defined statistically significant probabilities of p <0.05. At 6 months, the VAS CE group outperformed controls, but considering WMD and CI 95% values, and despite the out-of-significance range for the 6-month VAS UC group's p < 0.144, the CE and UC groups were likely comparable. The varied treatment and control group interventions limited the meta-analysis of Muthu et al. Only 3 out of the 17 pooled studies compared single intraarticular MSC-based injections to placebo injections, while 7 other studies compared MSC injections to controls that included PRP, hyaluronidase, and steroid injections. Further, 5 studies involved surgical procedures with adjuvant MSC therapy and interventional controls that were also surgical.

MSC heterogeneity encompasses multiple cell characteristics, including proliferation rate, immunophenotype, morphology, multilineage differential potential, and senescence[49]. Considering the proliferation potential of MSCs in culture, heterogeneity may produce small, round, and rapidly proliferating cells and large flattened cells that are slow to divide[50]. The number of colony-forming units (CFUs) consistently decreases with culture expansion, and CFU numbers become negligible after 20 passages[51]. To maximize the effectiveness of MSCs in clinical contexts, molecular aging markers are needed to assess for senescence-associated alterations in culture expansion. Morphologically, large-sized MSCs in culture with increased granularity coincide with cell senescence [52], and MSC autofluorescence has been put forth as a real-time, non-invasive, and quantifiable senescence marker[53]. Other senescence markers include -D-Galactosidase[54] (-Gal) and senescence-associated lysosomal -L-fucosidase (SA--Fuc). Since telomere length in cells decreases with each cell division until a critical telomere-length senescence stage occurs, they can be employed to predict senescence and estimate cellular division history[55]. The central focus now revolves around approaches that undo or mitigate tendencies toward senescence, which includes induced pluripotent stem cell-derived MSCs (IMSCs), genetic engineering that targets senescence-involved molecules, and pharmacologic approaches like rapamycin, which inhibits mechanistic target of rapamycin (mTOR).

The Blood-Brain Barrier

The blood-brain barrier (BBB) defines the network of endothelial cells that form the central nervous system's highly selective barrier, which 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 brain homeostasis and immune cell transport, and it is a crucial barrier protecting the CNS from toxins, pathogens, injury, inflammation, and disease[56]. The BBB's regulation comes from various physical, metabolic, and transport characteristics and its interactions with immune, neural, and vascular cells. This barrier may also prevent potential therapeutic drugs from entering the CNS meant to treat neurodegenerative diseases. Any compromise in the BBB's ability to maintain its selectiveness may allow pathogens to enter the CNS and cause neuroinflammation, contributing to neurodegenerative disease progression[57]. 

By default, a limited number of solutes can passively cross the BBB without the involvement of an active transport process. The shortlist of molecules that can passively diffuse through the BBB includes gases like oxygen and carbon dioxide, low molecular weight lipid-soluble molecules under 400 Da, or molecules containing less 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 transports to exert therapeutic benefits[59]. MSC homing may occur as a systemic or nonsystemic process. Nonsystemic homing represents a direct inoculation of MSCs into targeted tissues, where local cytokines guide them to injured tissues. In contrast, systemic homing refers to administering MSCs away from the injured tissues[60]. Several models have been proposed to describe the MSC homing mechanism, which involves MSCs leaving the circulation by endothelial integration, migration through the endothelial barrier, basement membrane penetration, and formation of plasmic podia to infiltrate the target tissues[61].  

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