Mitochondrial enrichment in infertile patients: a review of different mitochondrial replacement therapies

Poor ovarian responders exhibit a quantitative reduction in their follicular pool, and most cases are also associated with poor oocyte quality due to patient’s age, which leads to impaired in vitro fertilisation outcomes. In particular, poor oocyte quality has been related to mitochondrial dysfunction and/or low mitochondrial count as these organelles are crucial in many essential oocyte processes. Therefore, mitochondrial enrichment has been proposed as a potential therapy option in infertile patients to improve oocyte quality and subsequent in vitro fertilisation outcomes. Nowadays, different options are available for mitochondrial enrichment treatments that are encompassed in two main approaches: heterologous and autologous. In the heterologous approach, mitochondria come from an external source, which is an oocyte donor. These techniques include transferring either a portion of the donor’s oocyte cytoplasm to the recipient oocyte or nuclear material from the patient to the donor’s oocyte. In any case, this approach entails many ethical and safety concerns that mainly arise from the uncertain degree of mitochondrial heteroplasmy deriving from it. Thus the autologous approach is considered a suitable potential tool to improve oocyte quality by overcoming the heteroplasmy issue. Autologous mitochondrial transfer, however, has not yielded as many beneficial outcomes as initially expected. Proposed mitochondrial autologous sources include immature oocytes, granulosa cells, germline stem cells, and adipose-derived stem cells. Presently, it would seem that these autologous techniques do not improve clinical outcomes in human infertile patients. However, further trials still need to be performed to confirm these results. Besides these two main categories, new strategies have arisen for oocyte rejuvenation by improving patient’s own mitochondrial function and avoiding the unknown consequences of third-party genetic material. This is the case of antioxidants, which may enhance mitochondrial activity by counteracting and/or preventing oxidative stress damage. Among others, coenzyme-Q10 and melatonin have shown promising results in low-prognosis infertile patients, although further randomised clinical trials are still necessary.

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P–711 The association between thyroid autoimmunity and serum level of anti-Müllerian hormone in infertile patients with normal ovarian reserve undergoing in vitro fertilisation

Human Reproduction ◽

10.1093/humrep/deab130.710 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

A Albu ◽

D Albu

Keyword(s):

Serum Level ◽

Ovarian Reserve ◽

Thyroid Autoimmunity ◽

In Vitro Fertilisation ◽

Free T4 ◽

Infertile Women ◽

Group 2 ◽

Infertile Patients ◽

Group 1

Abstract Study question Is there a relationship between thyroid autoimmunity and serum level of anti-müllerian hormon (AMH) in infertile women with normal ovarian reserve undergoing in vitro fertilisation (IVF)? Summary answer In infertile women with normal ovarian reserve serum AMH level above 5ng/ml is associated with higher level of thyroid hormones and less frequent thyroid autoimmunity What is known already Previous studies suggest that thyroid autoimmunity is associated with a decreased ovarian reserve. Moreover, it was reported that thyroid hormone administration could improve serum AMH level. However, the relationship between serum AMH level and thyroid autoimmunity and function in infertile women with normal ovarian reserve undergoing IVF is largely unknown. Since in IVF the serum AMH level is an important marker which dictate the management of the couple, the identification of all the factors possibly related to this parameter is very important. Study design, size, duration: We performed a retrospective study in the Department of Reproductive Medicine of a private hospital. The medical records of all consecutive patients who underwent IVF between January 2015 and December 2018 with all causes of infertility were reviewed. Study group included 581patients with a mean age of 34.4±4.1 years, mean AMH of 3.78±2.4 ng/mL, mean serum TSH level of 1.89±1 microUI/ml and mean serum free T4 level of 1.05±0.98 ng/dl. Participants/materials, setting, methods Patients with known thyroid disorders or under thyroid hormone treatment at the moment of evaluation were excluded. Only patients with serum level of thyroid stimulating hormone (TSH), free tyroxine (free T4), anti thyroid peroxidase antibodies (ATPO,) anti thyroglobulin antibodies (ATG), AMH and age available for analysis were included in the study. This parameters are evaluated on a systematic basis in all the patients undergoing IVF in our Department, except very few cases. Main results and the role of chance Patients were divided according to their serum AMH level in two groups: group 1 with AMH level 5 ng/ml and below (n = 450 patients) and group 2 with AMH above 5 ng/ml (n = 131 patients). When the two groups were compared we found that patients in group 2 were younger in comparison with patients in group 1 (32.9±3.8 versus 35±4 years, p < 0.0001). After adjustment for age, patients in group 2 had significantly higher serum free T4 level (1.07±0.12 versus 1.04±0.14 ng/dl, p = 0.015), lower ATG (17.59±41.8 UI/ml versus 39.4±136.16 UI/ml, p < 0.018) and presented less frequently with high ATPO antibodies (35% versus 41.8%, p = 0.047). In a logistic regression model with AMH as a dependent variable, free T4, but not TSH was independently and positively associated with higher AMH levels (above 5 ng/ml) (p = 0.025) after adjustment for anti thyroid antibodies levels. Morever, in this logistic model the presence of high ATPO, but not ATG, were negatively related to higher AMH level (p = 0.037). Limitations, reasons for caution Patients included in this study are infertile patients with indication for IVF treatment. Therefore, the results of this study should be used with caution in other populations Wider implications of the findings: Our study suggest that serum AMH level might be related to thyroid autoimmunity, but also to thyroid hormones levels. If confirmed by further studies, this findings could offer a way to improve serum AMH level and to better understand the markers of ovarian reserve in an IVF setting. Trial registration number NA

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253 OXIDATIVE STRESS MAY IMPAIR OOCYTE QUALITY IN DAIRY COWS OF REPRODUCTIVE AGE WITH A REDUCED ANTRAL FOLLICLE COUNT

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab253 ◽

2013 ◽

Vol 25 (1) ◽

pp. 274 ◽

Cited By ~ 1

Author(s):

I. Tessaro ◽

F. Franciosi ◽

V. Lodde ◽

D. Corbani ◽

A. M. Luciano ◽

...

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Antral Follicle ◽

Oocyte Quality ◽

Antral Follicle Count ◽

Oxide System ◽

Reproductive Age ◽

Developmental Competence ◽

Mitochondrial Activity

In dairy cattle, oocytes isolated from ovaries with a reduced antral follicle count (AFC) have a low embryonic developmental competence. This may be related to oxidative stress, as indicated by our recent finding that ovaries with reduced AFC show a defective endothelial nitric oxide synthase/nitric oxide system. To further test this hypothesis, we evaluated whether the poor developmental competence of these oocytes was possibly due 1) to an imbalance of the reduced glutathione (GSH) system, because GSH is the major antioxidant compound stored within the oocyte and protects the zygote and early embryos from oxidative damage, and 2) to reduced mitochondrial activity. Ovaries were obtained from the abattoir, and oocytes were collected from ovaries with reduced AFC, with fewer than 10 follicles of 2 to 6 mm in diameter, and aged-matched controls, with more than 10 follicles of 2 to 6 mm in diameter. Oocyte GSH content was evaluated using the 5,5′-dithio-bis(2-nitrobenzoic acid)-GSH reductase recycling micro-GSH assay before and after in vitro maturation (IVM) in the presence or absence of 100 µM cysteamine, a GSH precursor. At the same time the developmental competence after IVF was assessed. Moreover, the mitochondrial activity during IVM was evaluated in additional oocytes from the two ovarian categories by specific MitoTracker dyes (MitoTracker FM Green and MitoTracker Orange CMTMRos, Invitrogen, Carlsbad, CA, USA) and subsequent image analysis (ImageJ software). All data were analysed by ANOVA followed by Fisher’s least significant differences test, and P-values <0.05 were considered significant. Experiments were repeated at least three times. Oocytes isolated from ovaries with a low AFC had a similar GSH content compared with oocytes isolated from control ovaries (n = 65 and 85, respectively; 4.31 ± 0.41 v. 4.51 ± 0.42 pmol oocyte–1). After IVM, oocytes from ovaries with reduced AFC showed a significantly lower GSH content compared with control oocytes (n = 55 and 65, respectively; 4.36 ± 0.31 v. 6.59 ± 0.39 pmol oocyte–1); however, cysteamine supplementation during IVM induced GSH accumulation similar to the control (n = 80 and 85, respectively; 9.88 ± 0.77 v. 10.45 ± 0.88 pmol oocyte–1). It is interesting that the increase in intracellular GSH content significantly improved the developmental competence of oocytes from ovaries with a reduced AFC (n = 196 and 201, respectively; 20.1 ± 2.9% v. 6.2 ± 1.6%), although the blastocyst rate remained lower than the control either with or without cysteamine (n = 218 and 212, respectively; 33.3 ± 3.8% and 34.2 ± 2.4%). Further, immature oocytes from ovaries with a low AFC showed a reduced mitochondrial membrane potential compared with control oocytes (n = 13 and 18, respectively; 1.74 ± 1.19 v. 2.22 ± 1.72, calculated as the ratio between the fluorescence of active and total mitochondria), whereas at the end of IVM, it declined in both categories at a comparable level (n = 17 and 24, respectively; 1.19 ± 0.10 and 1.30 ± 0.06). Our data confirmed the hypothesis that both the GSH imbalance and defective mitochondrial activity contribute to the limited developmental competence of oocytes from ovaries with a reduced AFC. This work was supported by Dote ricerca applicata-FSE, Regione Lombardia, Italy (VL, IT).

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Human Mesenchymal Stem Cells Inhibit In Vitro Antibody Production Against Alloantigens.

Blood ◽

10.1182/blood.v106.11.3033.3033 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3033-3033

Author(s):

Patrizia Comoli ◽

Rita Maccario ◽

Maria Antonietta Avanzini ◽

Fabrizio Ginevri ◽

Antonia Moretta ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

B Cell ◽

Antibody Production ◽

Third Party ◽

Favourable Effect ◽

Solid Organ ◽

Hematopoietic Stem ◽

Cross Match

Abstract Antibodies directed against alloantigens are implicated in the pathogenesis of several immune reactions complicating transplantation. In particular, this humoral response unfavorably affects the outcome of solid organ transplantation, and it has been hypothesized to be responsible for some of the clinical manifestations related to graft-versus-host disease (GVHD). In detail, the presence of antibodies against donor cells is a contraindication to kidney transplantation because of the risk of hyperacute rejection. In the effort to expand the donor pool, trials of allograft transplantation across HLA-sensitization have been conducted by means of strategies including pre-transplant plasmapheresis, intravenous immunoglobulins (Ig), anti-B cell monoclonal antibodies and splenectomy, associated with high-intensity immunosuppressive regimens. These measures have proved only partially successful in preventing humoral rejection in high-risk patients. Thus, the development of new therapeutic tools able to blunt alloantibody production could be a welcomed implementation to existing protocols. Mesenchymal stem cells (MSC) have been demonstrated to possess immunomodulatory capacity, since they induce T-cell hyporesponsiveness in vitro, prolong survival of skin graft in a primate model, and seem to decrease GVHD incidence and severity in humans given hematopoietic stem cell transplantation. To verify whether MSC may exert an inhibitory effect on antibody production, we stimulated B-cell-enriched peripheral blood mononuclear cells (PBMC) obtained from healthy controls (n=9) or sensitized prospective kidney recipients (n=5) in a mixed lymphocyte culture (MLC) against irradiated HLA-disparate stimulator PBMC (controls) or stimulators cells bearing HLA antigens matched with the positive cross-match (patients). Antibody production in the absence or in the presence of third-party allogeneic MSC (responder:MSC ratio:4:1) was then evaluated by ELISA. We found that the addition of MSC at the beginning of MLC considerably inhibited IgG and IgM production (median fold-decrease of IgG production: controls, 7; patients, 5; median fold-decrease of IgM production: controls, 17; patients, 4). Our preliminary findings indicate that third-party MSC are able to suppress antibody production in vitro, and may therefore help to overcome a positive cross-match in sensitized transplant recipients. These results may also contribute to partly explain the mechanism at the basis of the favourable effect played by MSC in patients with GVHD.

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Mesenchymal Stem Cells for Treatment of Severe Acute Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v108.11.5304.5304 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5304-5304 ◽

Cited By ~ 6

Author(s):

Katarina Le Blanc ◽

Francesco Frassoni ◽

Lynne Ball ◽

Edoardo Lanino ◽

Berit Sundberg ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Graft Versus Host Disease ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Third Party ◽

Host Disease ◽

Graft Versus Host ◽

Acute Graft

Abstract Mesenchymal stem cells (MSC) from adult bone marrow have the capacity to differentiate into several mesenchymal tissues and inhibit T-cell alloreactivity in vitro. Within the EBMT MSC expansion consortium we have used MSC to treat grades III–IV acute graft-versus-host disease (GvHD) in 40 patients. The MSC dose was median 1.0 (range 0.4–9) 10^6 cells/kg body weight of the recipient. No side-effects were seen after MSC infusions. Nineteen patients received one dose, 19 patients received two doses, two patients received three and five doses respectively. MSC donors were in five cases HLA-identical sibling donors, 19 haploidentical donors and 41 third-party HLA-mismatched donors. Among the 40 patients treated for severe acute GvHD, 19 had complete responses, nine showed improvement, seven patients did not respond, four had stable disease and one patient was not evaluated due to short follow-up. Twenty-one patients are alive between six weeks up to 3.5 years after transplantation. Nine of these patients have extensive chronic GvHD. One patient with ALL has recurrent leukaemia and one patient has denovo AML of recipient origin. We conclude that MSC have immunomodulatory and tissue repairing effects and should be further explored as treatment of severe acute GvHD in prospective randomized trials.

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Dietary carbohydrates and amino acids influence oocyte quality in dairy heifers

Reproduction Fertility and Development ◽

10.1071/rd08193 ◽

2009 ◽

Vol 21 (3) ◽

pp. 419 ◽

Cited By ~ 14

Author(s):

J. A. Rooke ◽

A. Ainslie ◽

R. G. Watt ◽

F. M. Alink ◽

T. G. McEvoy ◽

...

Keyword(s):

Amino Acids ◽

Plasma Insulin ◽

Antral Follicle ◽

Oocyte Quality ◽

High Plasma ◽

In Vitro Fertilisation ◽

Dairy Heifers ◽

High Starch ◽

Starch Diet

The objective of the present experiment was to determine whether increasing plasma insulin by different nutritional regimes affects oocyte quality. Holstein dairy heifers (eight per treatment) were assigned, using a two times two factorial design, to diets containing either low or high dietary leucine and either low or high dietary starch. Each heifer underwent six sessions of ovum pick-up beginning 25 days after introduction of the diets. Oocyte quality was assessed by development to the blastocyst stage in synthetic oviducal fluid following in vitro fertilisation. Feeding diets containing high leucine resulted in significantly higher plasma free leucine and tyrosine concentrations. The high-starch diet significantly increased plasma insulin but not glucagon concentration, whereas high dietary leucine increased plasma glucagon but not insulin. Oocyte cleavage was not influenced by diet. The high-starch diet, which was associated with a high plasma insulin : glucagon ratio, had adverse effects on oocyte quality that were avoided when leucine intake was increased. There was an association between total plasma free amino acid concentration and oocyte cleavage. Therefore, in dairy heifers dietary amino acids and carbohydrates during antral follicle development appear to mediate effects on oocyte quality by different mechanisms. These findings have implications for both diet formulation and feeding regimes.

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Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200502073 ◽

2005 ◽

Vol 170 (5) ◽

pp. 721-732 ◽

Cited By ~ 147

Author(s):

Phillip Karpowicz ◽

Cindi Morshead ◽

Angela Kam ◽

Eric Jervis ◽

John Ramunas ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Genetic Material ◽

Precursor Cells ◽

Neural Precursor Cells ◽

Dna Templates ◽

Cell Divisions ◽

Immortal Strand ◽

Asymmetric Partitioning

The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

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Mitochondrial activity of cancer and normal mesenchymal stem cells in vitro cultured in medium with different deuterium content

BIO Web of Conferences ◽

10.1051/bioconf/20202202005 ◽

2020 ◽

Vol 22 ◽

pp. 02005

Author(s):

Igor Zlatskiy ◽

Nadine Antipova ◽

Alona Zlatska ◽

Svitlana Dolenko ◽

Anton Syroeshkin

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Growth Medium ◽

Diffraction Method ◽

Laser Diffraction ◽

Mitochondrial Activity ◽

Deuterium Content ◽

Correlation Dependence ◽

Density Inhomogeneities

We showed that cancer and normal mesenchymal stem/stromal (MSC) cells in vitro in a deuterated growth medium show a decrease of mitochondrial activity (MA), while in a deuterium-depleted medium an increase. This was established using mitotracker and rhodamine 123, and was also confirmed by expression of the UCP1 gene. The correlation dependence of mitochondrial activity in a medium with a changed ratio of deuterium/protium (D/H) and supramolecular structures was established, using the laser diffraction method. Density inhomogeneities in the deuterated medium are noted to be large, and in the deuterium-depleted medium small, in comparison with the control.

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Abstract 14859: Mesenchymal Stem Cells Rescue Patient-Specific Cardiomyocyte Viability and Function Following Doxorubicin Injury via Microvesicle Mediated Mitochondrial Transfer to Recapitulate Human Clinical Trial Results

Circulation ◽

10.1161/circ.142.suppl_3.14859 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Connor G OBrien ◽

Mehmet O Ozen ◽

Evgeniya Vaskova ◽

Jihye Jung ◽

Michelle Santoso ◽

...

Keyword(s):

Clinical Trial ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Treated Patient ◽

Chemotherapeutic Agents ◽

Patient Specific ◽

P Value ◽

Mitochondrial Transfer ◽

And Function

Introduction: Anthracyclines are effective chemotherapeutic agents associated with cardiac toxicity. Anthracycline induced cardiomyopathy (AIC) carries a mortality of 20% at four years. Effective therapies for AIC have not been established. The NIH-sponsored Stem Cell Injection for Cancer Survivors (SENECA) Trial examined the role of allogeneic mesenchymal stem cells (MSC) to treat AIC. To predict the efficacy of MSC in the SENECA patients, we performed an in vitro clinical trial to study the restorative effects of MSC on patient-specific iPSC-derived cardiomyocytes (iCM) by assessing the effects in vitro following doxorubicin (dox) exposure. Hypothesis: MSC secretome will rescue SENECA Trial patient-specific iCM from dox injury. Methods and Results: We generated three SENECA patient-specific iPSC lines. In vitro iCM dox sensitivity was shown to be comparable to published values. iCM were cultured in 1μM dox for 24hrs, treated with MSC or MSC-secretome, and assessed for viability and apoptosis. Co-culture of MSC with iCM using a 1μm trans-well system improved iCM viability by 43% (p-value < 0.005) and reduced apoptosis (p-value = 0.013). To better define the paracrine mechanism, MSC-secretome was divided into microvesicles >200nm (MV) and exosomes <200nm (Ex) using a novel filtration system. MV recapitulated the effect of co-culture, improving iCM viability by 87.8% (p-value = 0.006) and reducing apoptosis by 60% (p-value = 0.013). Treatment with Ex had no effect. MV restore contractility and calcium transients in the dox-injured iCM. MV were found to contain mitochondria (MT), which are taken up by iCM. Treated iCM demonstrate increased MT-copy number and augmented MT-biogenesis. Inhibition of MT function in MV attenuated therapeutic efficacy. We were unblinded to SENECA outcomes. In our cohort, the MSC-treated patient improved across all endpoints while placebo treated patients did not consistent with our in vitro findings. Conclusion: Allogeneic MSC attenuate apoptosis and restore contractility following dox injury in patient-specific iCM. MV and MT-transfer appear to be the putative mechanism. Our findings correlate with the SENECA results, demonstrating proof-of-concept that iPSC-derivatives can be used to predict trial outcomes.

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Stem cells and organs-on-chips: new promising technologies for human infertility treatment

Endocrine Reviews ◽

10.1210/endrev/bnab047 ◽

2021 ◽

Author(s):

Eisa Tahmasbpour Marzouni ◽

Andrew Henrik Sinclair ◽

Catharyn Stern ◽

Elena Jane Tucker

Keyword(s):

Stem Cells ◽

Primordial Germ Cell ◽

Reproductive Failure ◽

Human Reproduction ◽

Patient Specific ◽

Sex Development ◽

Organ On A Chip ◽

Induced Pluripotent ◽

Infertile Patients

Abstract Having biological children remains an unattainable dream for most couples with reproductive failure or gonadal dysgenesis. The combination of stem cells with gene editing technology and organ-on-a-chip models provides unique opportunity for infertile patients with impaired gametogenesis caused by congenital disorders in sex development or cancer survivors. But, how will these technologies overcome human infertility? This review discusses the regenerative mechanisms, applications and advantages of different types of stem cells for restoring gametogenesis in infertile patients, as well as major challenges that must be overcome prior to clinical application. The importance and limitations of in vitro generation of gametes from patient-specific human induced pluripotent stem cells (hiPSCs) will be discussed in the context of human reproduction. The potential role of organ-on-a-chip models that can direct differentiation of hiPSCs-derived primordial germ cell-like cells to gametes and other reproductive organoids is also explored. These rapidly evolving technologies provide future prospects for improving fertility to individuals and couples who experience reproductive failure.

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Mesenchymal Stem Cells Induce a Reversible Suppression of Alloimmune Reactions.

Blood ◽

10.1182/blood.v106.11.4316.4316 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4316-4316

Author(s):

Sabine Tschiedel ◽

Kristina Bartsch ◽

Annette Reinhardt ◽

Gentilini Chiara ◽

Niederwieser Dietger

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Ex Vivo ◽

Primary Response ◽

Significant Inhibition ◽

Third Party ◽

Loss Of Function ◽

Inhibition Of Proliferation

Abstract Objective Bone marrow contains pluripotent mesenchymal stem cells (MSC) that form bone, cartilage, adipose tissue and muscle. These cells are not immunogenic and escape recognition by alloreactive T cells and natural killer cells. MSC can be readily isolated from bone marrow and expanded ex vivo without modification in phenotype or loss of function. They are capable of differentiating along multiple mesenchymal lineages, play a crucial role in the bone marrow microenvironment and have profound immunosuppressive properties. In the present investigation we analysed the antiproliferative capacity of MSC using primary and secondary immune responses in vitro. Methods MSC were isolated from heparinized bone marrow of healthy donors (n=5). Bone marrow aspirates were layered onto a Percoll cushion (density 1,073g/ml) and the MSC-enriched mononuclear fraction was collected, washed and resuspended in Dulbecco modified Eagle medium with low glucose and 10% fetal bovine serum. The cells were plated at 2x105/cm2 and maintained at 37°C in a humidified atmosphere and subcultured prior to confluency. MSC stained positive for specific markers as SH2, SH3 and SH4 but were negative for CD14, CD34 and CD45. Immunsuppressive effects were assessed by adding third party MSC to primary mixed lymphocyte reactions (MLR) in decreasing concentrations (10%, 1%, 0,1%) on days 0–5. Cell proliferation was measured on day 6 by means of an 18-hour pulse with 3H-thymidine (3H-TdR). Secondary MLRs were performed by restimulating MSC co-cultured MLRs with the primary PBMC on day 10. In addition secondary MLRs were also tested in presence of MSC and cultured for 1, 2, 3 and 6 days in 96-well plates and proliferation was detected by 3H-TdR uptake. Results Primary MLRs show significant inhibition of proliferation with 10% MSC added on day 0 (85% inhibition), 1 (69% inhibition) or 2 (70% inhibition) (p<0,01). Similar effects were observed by adding 1% MSC on day 0 (42% inhibition) and 1 (10% inhibition). Co-cultivation with MSC over 10 days lead to delayed proliferation in secondary MLRs with peak on day 3. Secondary MLRs were also inhibited by the addition of 1% (47% inhibition) and 10% (56% inhibition) MSC independent from co-cultivation with MSC in the primary culture (11% and 44% inhibition respectively). Restimulation with third party PBMC on day 10 lead to a primary response with a proliferation peak after 6 days. Conclusions Our findings emphasize the immunosuppressive effects of MSC. We observed that MSC-induced inhibition of primary MLRs was reversible in secondary MLRs. This clearly demonstrates that MSC do not induce tolerance and could be used in a clinical setting due to their immunomodulatory properties.

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