Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture

Objectives: This study has aimed to repopulate ‘primitive’ cells from late‐passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Materials and methods: Effects of low density culture compared t high density culture on late‐passage bone marrow (BM)‐derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. Results and conclusions: We repopulated ‘primitive’ cells by replating late‐passage MSCs at low density (17 cells/cm²) regardless of donor age. Repopulated MSCs from low‐density culture were smaller cells with spindle shaped morphology compared to MSCs from high‐density culture. The latter had enhanced colony‐forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic‐related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late‐passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct‐4), Osterix and Msx2 reverted to levels of early‐passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony‐forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of ‘primitive’ cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2.     

Construction and characterization of osteogenic and vascular endothelial cell sheets from rat adipose-derived mesenchymal stem cells

In this study, adipose-derived mesenchymal stem cells (ADSCs) were isolated from adipose tissues of rats. Flow cytometry identification showed that ADSCs of passage 3 highly expressed CD29 and CD44, but hardly expressed CD31 and CD45. Adipogenic, osteogenic, and chondrogenic differentiation were confirmed by the results of oil red O staining, alkaline phosphatase (ALP), and alcian blue staining, respectively. ADSCs at a density of 1 × 10⁶/cm² were cultured in the osteogenic medium and the osteogenic cell sheets could be obtained after 14 d. The cell sheets were positive with von kossa staining. The transmission electron microscopy (TEM) result showed that needle-like calcium salt crystals were deposited on the ECM. These results suggested that the osteogenic cell sheets may have potential osteogenesis ability. ADSCs at a density of 1 × 10⁶/cm² were cultured in the endothelial cell growth medium-2 and the endothelial cell sheets can be formed after 16 d of culture. The TEM image confirmed that the Weibel-Palade corpuscle was seen in the cells. The expression of CD31 was positive, suggesting that the endothelial cell sheets may have a strong ability to form blood vessels. In this study, two types of cell sheets with the potential abilities of osteogenesis and blood vessels formation were obtained by induced culture of ADSCs in vitro, which lays a foundation to build vascularized tissue engineered bone for the therapy of bone defects.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Cell growth and nutritive value of the tropical benthic diatom, <Emphasis Type="Italic">Amphora</Emphasis> sp., at varying levels of nutrients and light intensity,and different culture locations

Two series of experiments were conducted to determine suitable growth factors for the mass propagation of the local algal isolate Amphora sp. A higher growth rate of 0.2 doubling (μ) day⁻¹ was attained at a lower photosynthetic photon flux density (PPFD; 11.4 μmol photon m⁻²s⁻¹) compared to cultures exposed to higher levels of PPFD (16.1 μmol photon m⁻²s⁻¹, −0.1 μ day ⁻¹; 31.3 μmol photon m⁻²s⁻¹, 0.0 μ day⁻¹). Cultures located inside the laboratory had a significantly higher cell density (133 × 10⁴ cells cm⁻²) and growth rate (0.3 μ day⁻¹) compared to those located outdoors (100 × 10⁴ cells cm⁻², 0.2 μ day⁻¹). A comparison of nutrient medium across two locations showed that lipid content was significantly higher in cultures enriched with F/2MTM (macronutrients + trace metals) and F/2MV (macronutrients + vitamins). Saturated fatty acids were also present in high concentrations in cultures enriched with F/2M (macronutrients only). Significantly higher amounts of saturated fatty acids were observed in cultures located outdoors (33.1%) compared to those located indoors (26.6%). The protein, carbohydrates and n-6 fatty acid content of Amphora sp. were influenced by the location and enrichment of the cultures. This study has identified growth conditions for mass culture of Amphora sp. and determined biochemical composition under those culture conditions. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Correlation between cell attachment areas after 2 h of culture and osteogenic differentiation activity of rat mesenchymal stem cells on hydroxyapatite substrates with various surface properties

The initial attachment of mesenchymal stem cells (MSCs) to substrates and osteogenic differentiation are supported by culture on a hydroxyapatite substrate. Cell attachment areas of rat MSCs after 2 h of culture on hydroxyapatite substrates with various microstructures and the osteogenic differentiation activity thereafter were measured. The perceived outcome was that, after 2 h of culture, rat MSCs with a small attachment area would have a high osteogenic differentiation activity, whereas those with a large attachment area would have a low osteogenic differentiation activity. Furthermore, rat MSCs with a small attachment area had many cytoplasmic processes, while those with a large attachment area revealed clear stress fibers and focal contacts. These results suggest that cell attachment area of rat MSCs after 2 h of culture has a strong effect on the osteogenic differentiation of rat MSCs. Thus, the measurement of cell attachment area after 2 h of culture could become valuable for estimating the osteogenic differentiation activity of rat MSCs thereafter.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

A non-contact suspension culture approach to the culture of osteogenic cells derived from a CD49elow subpopulation of human bone marrow-derived cells

We demonstrate that adult human bone marrow (BM) contains a population of mesenchymal stromal cells (MSCs) that can be expanded in non-adherent, cytokine-dependent, suspension culture conditions for at least 42 days. The cells generated during suspension culture lacked detectable levels of gene expression associated with differentiated mesenchymal cell types, including bone, muscle and fat, suggesting that suspension culture maintains MSCs in an uncommitted state. However, when these undifferentiated cells were taken out of suspension culture and placed in adherent osteogenic conditions, osteogenic genes were upregulated and morphologically identifiable bone matrix was elaborated. Flow cytometric analysis of uncultured, density gradient-separated human BM revealed that colony forming unit-fibroblast (CFU-F) and CFU-osteoblast (CFU-O) activity was associated with a CD45(-) CD49e(low) phenotype. Importantly, suspension-grown MSCs, capable of CFU-F and CFU-O development, maintained the CD45(-)CD49e(low) phenotype whereas MSCs directly cultured under adherent conditions rapidly upregulated CD49e expression and were associated with a CD45(-)CD49e(high) phenotype. Tracking the CD49e(low) expression under suspension culture conditions provides a mechanism to isolate an expanding suspension-grown MSC population with osteogenic potential. This could provide a potential strategy to isolate populations of MSCs, with functional osteogenic capacity, in a scalable and controllable culture system for therapeutic applications.     

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b⁻ cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b⁻ cells increased the number of AlkP⁺ cells from the plastic adherent fraction from 6.3% ± 0.8 to 56% ± 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b⁻ fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.     

Kinetic models for phycocyanin production by high cell density mixotrophic culture of the microalga Spirulina platensis

Phycocyanin production by high cell density cultivation of Spirulina platensis in batch and fed-batch modes in 3.7-L bioreactors with a programmed stepwise increase in light intensity program was investigated. The results showed that the cell density in fed-batch culture (10.2 g L⁻¹) was 4.29-fold that in batch culture (2.38 g L⁻¹), and the total phycocyanin production in the fed-batch culture (0.795 g L⁻¹) was 3.05-fold that in the batch culture (0.261 g L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, phycocyanin formation, as well as glucose consumption was proposed. The data fitted the models well (r ² > 0.99). Furthermore, based on the kinetic models, the potential effects of light limitation and photoinhibition on cell growth and phycocyanin formation can be examined in depth. The models demonstrated that the optimal light intensity for mixotrophic growth of Spirulina platensis in batch or fed-batch cultures using a 3.7-L bioreactor was 80160 μE m⁻² s⁻¹, and the stepwise increase in light intensity can be replaced by a constant light intensity mode. Received 28 July 1998/ Accepted in revised form 8 October 1998     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

Effects of microgravity simulation on the production of interleukins in culture of human mesenchymal stromal cells

The effects of microgravity that were simulated using clinoritation and cell exposure in a Random Positioning Machine (RPM; Dutch Space, Netherlands) on the production of interleukins by mesenchymal stromal cells (MSCs) of the human bone marrow were studied. Additionally, interleukins production was studied in osteogenic derivatives of MSCs induced by growth factors, such as 10⁻⁸ M dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerolphosphate. Twenty-day clinoritation resulted in an increase in the interleukin-8 (IL-8) level by a factor of 1.4–3.2 in the culture medium of MSCs and their osteogenic derivatives. Exposure of cells to RPM was followed by an increase in IL-8 production by factors of 1.5–6 and 1.6–2.1 after 10 and 20 days of the treatment, respectively. MSCs and their osteogenic derivatives exhibited a tendency towards a decrease in the release of IL-6 into the culture medium after simulation of microgravity with RPM. Thus, in different models, microgravity modified production of interleukins in MSCs and mature cells with the osteogenic phenotype.     

Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment

Summary Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for cell attachment. PUF has a macroporous structure giving a high surface area to volume ratio. Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of PUF and grew to a high cell density (1.04 × 10⁸ cells/ cm³ PUF and 3.5 × 10⁷ cells/ cm³ PUF, respectively) in PUF stationary cultures. In addition, we have designed a PUF-particle packed-bed culture system for high density mass cell culture. A maximum cell density of 2.4 × 10⁷ cells/cm³ culture vessel volume was obtained in a packed-bed culture of Vero cells. Offprint requests to: K. Funatsu     

Optimization of culture conditions for tissue culture production of young plantlets of carrageenophyte <Emphasis Type="Italic">Kappaphycus</Emphasis>

To improve the production of Kappaphycus plantlets in tissue culture, optimum media concentrations of an Ascophyllum nodosum extract (Acadian Marine Plant Extract Powder, AMPEP), plant growth regulators (PGR), pH–temperature combinations, and explant density were determined. Kappaphycus alvarezii var. tambalang purple (PUR), kapilaran brown (KAP), vanguard brown (VAN), adik-adik (AA), tungawan green (TGR), and K. striatum var. sacol green (GS) were used as explants. Based on the shortest period for shoot emergence and the economical use of AMPEP, the optimum enriched media was 3.0 mg L⁻¹ AMPEP and 0.1 mg L⁻¹ AMPEP + PGR 1 mg L⁻¹ each phenylacetic acid (PAA) and zeatin for PUR, 1.0 mg L⁻¹ AMPEP + PGR for KAP and GS, 0.1 mg L⁻¹ AMPEP + PGR for VAN, and 3.0 mg L⁻¹ AMPEP and 0.001 mg L⁻¹ AMPEP + PGR for AA and TGR. Results showed that the addition of PGR to low concentrations of AMPEP hastened shoot formation. pH–temperature combinations for the most rapid shoot formation were determined for the brown (KAP) and purple (PUR) color morphotypes of K. alvarezii var. tambalang and the green morphotype of K. striatum var. sacol (GS) cultured in 1.0 mg L⁻¹ AMPEP + PGR. The brown morphotype produced the most number of shoots at pH 7.7 at 20°C after as little as 20 days. Purple K. alvarezii showed an increased shoot formation at pH 6.7 at 25°C and the green K. striatum morphotype at pH 8.7 at 25°C. The optimum number of explants added to the culture media was also determined for tungawan green (TGR), brown (KAP), and tambalang purple (PUR) varieties of K. alvarezii in 1.0 mg L⁻¹ AMPEP + PGR. The number of explants and the volume of the culture media combination were also tested. The highest average number of shoots formed occurred in two explants:1 mL culture media (2:1) for KAP and PUR (35.00% and 16.67%, respectively) and 1 explant: 2 mL culture media for the TGR (100.00%) with a range of 0.5–3.0 mm shoot length after 40 days in culture. The earliest shoot formation was observed after 21 days for the brown and 9 days for both the green and purple color morphotypes of Kappaphycus, in all densities investigated. This indicated that within the range tested, the density of explants did not have a significant effect on the rate of shoot formation but did influence the average number generated from the culture. The rate of production of new and improved Kappaphycus explants for a commercial nursery stock was improved through the use of AMPEP with optimized culture media pH, temperature, and density conditions.     

High density culture of anchorage-dependent animal cells by polyurethane foam packed-bed culture systems

Summary Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of polyurethane foam (PUF) and grew to a high cell density (1.1 × 10⁸ cells/cm³ PUF and 4.2 × 10⁷ cells/cm³ PUF, respectively) in a PUF-plates packed-bed culture system. This density of Vero cells was twice that obtained previously with a PUF-particles packed-bed culture system. A maximum cell density of 6.7 × 10⁷ cells/cm³ culture vessel volume was obtained in a PUF-disc packed-bed culture of Vero cells. From the cell density of CHO-K1, growing in a monolayer on the surface of PUF and a petri dish, per bulk volume of PUF, we estimated that a surface area to volume ratio of PUF plates effective for cell growth was about 109 cm²/cm³.Offprint requests to: K. Funatsu     

Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche

In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a “biomimetic niche.” The CD34⁺ cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2–5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34⁺ cells from umbilical cord blood in the 3D system increased 3.3–4.8 folds when compared with the initial CD34⁺ cells. CD34⁺/CD38⁻ cells accounted for 82–90% of CD34⁺ cells. After 5 weeks, CD34⁺/CD38⁻ cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10³ vs. 1.0 ± 0.5 × 10⁴, p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10³ vs. 2.5 ± 0.7 × 10², p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6–9.3 folds vs. 1.0–1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2–7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.     

Tissue plasminogen activator (t-PA) production by a high density culture of weakly adherent human embryonic kidney cells using a polyurethane-foam packed-bed culture system

Weakly adherent cells of the 293 line attached well to the internal surface of polyurethane foam (PUF) and grew to the high density of 6.83 × 10⁷ cells/cm³ PUF in stationary culture. The maximum productivity of tissue plasminogen activator (t-PA) was 0.158 IU/10⁶ cells per day. The productivity decreased at the stationary phase of cell growth, so we designed a PUF-plate packed-bed culture system for high density culture and continuous production of t-PA. A maximal cell density of 3.24 × 10⁷ cells/cm³ PUF and a t-PA productivity of 0.326 IU/10⁶cells per day were obtained in 25-day perfusion cultures. Although the cell density decreased to half that in PUF stationary culture, the t-PA productivity increased twofold and was maintained for 25 culture days.     

Cell culture density affects the proliferation activity of human adipose tissue stem cells

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT‐MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT‐MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm^?2. After 7 days of incubation, P4 and P12 AT‐MSCs cultured in CC1 were thin and spindle‐shaped, whereas those cultured in CC2 had extensive cell‐to‐cell contacts and an expanded cell volume. In addition, P4 and P12 AT‐MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)‐carboxyfluorescein diacetate N‐succinimidyl ester dye showed that the fluorescence intensity of AT‐MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation‐associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT‐MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT‐MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

Substance P enhances mesenchymal stem cells-mediated immune modulation

Since clinical application of MSCs requires long-term ex vivo culture inducing senescence in MSCs and reducing the therapeutic activity of transplanted MSCs, numerous efforts have been attempted to sustain the active state of MSCs. Substance P (SP) is a neuropeptide that functions to activate the cellular physiological responses of MSCs, including proliferation, migration, and secretion of specific cytokines. In this study, we explored the potential of SP to restore the weakened immune modulating activity of MSCs resulting from long-term culture by measuring T cell activity and interleukin-2 (IL-2) secretion of CD4⁺ Jurkat leukemic T cells and primary CD4⁺ T cells. As the number of cell passages increased, the immunosuppressive function of MSCs based on T cell activity decreased. This weakened activity of MSCs could be restored by SP treatment and nullified by co-treatment of an NK1 receptor blocker. Higher levels of transforming growth factor beta 1 (TGF-β1) secretion were noted in the medium of SP-treated late passage MSC cultures, but IL-10 levels did not change. SP-treated MSC-conditioned medium decreased T cell activity and IL-2/Interferon gamma (IFN-g) secretion in T cells even in the activation by lipopolysaccharide (LPS) or CD3/CD28 antibodies, both of which were successfully blocked by inhibiting the TGF beta signaling pathway. This stimulatory effect of SP on late passage MSCs was also confirmed in direct cell–cell contact co-culture of MSCs and CD4⁺ Jurkat T cells. Collectively, our study suggests that SP pretreatment to MSCs may recover the immunosuppressive function of late passage MSCs by potentiating their ability to secrete TGF-β1, which can enhance the therapeutic activity of ex vivo expanded MSCs in long-term culture.