Limits...
A robust potency assay highlights significant donor variation of human mesenchymal stem/progenitor cell immune modulatory capacity and extended radio-resistance.

Ketterl N, Brachtl G, Schuh C, Bieback K, Schallmoser K, Reinisch A, Strunk D - Stem Cell Res Ther (2015)

Bottom Line: Reports on functional heterogeneity of MSPCs created additional uncertainty regarding donor and organ/source selection.Comparing various MSPCs revealed significant potency inconsistency and generally diminished allo-immunosuppression compared to dose-dependent inhibition of mitogenesis.Gamma-irradiation to block unintended MSPC proliferation did not prohibit chondrogenesis and osteogenesis in vivo, indicating the need for alternative safety strategies.

View Article: PubMed Central - PubMed

Affiliation: Experimental and Clinical Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center, Paracelsus Medical University, Salzburg, Austria. nina.ketterl@pmu.ac.at.

ABSTRACT
The inherent immunomodulatory capacity of mesenchymal stem/progenitor cells (MSPCs) encouraged initiation of multiple clinical trials. Release criteria for therapeutic MSPCs cover identity, purity and safety but appropriate potency assessment is often missing. Reports on functional heterogeneity of MSPCs created additional uncertainty regarding donor and organ/source selection. We established a robust immunomodulation potency assay based on pooling responder leukocytes to minimize individual immune response variability. Comparing various MSPCs revealed significant potency inconsistency and generally diminished allo-immunosuppression compared to dose-dependent inhibition of mitogenesis. Gamma-irradiation to block unintended MSPC proliferation did not prohibit chondrogenesis and osteogenesis in vivo, indicating the need for alternative safety strategies.

Show MeSH

Related in: MedlinePlus

Irradiated MSPC maintain their immunomodulatory potency in vitro and their differentiation capacity in vivo. a Direct comparison of the inhibition of phytohemagglutinin (PHA)-induced T-cell proliferation (green bar) by non-irradiated bone marrow (BM)-MSPCs (-Rx; blue bars) versus 30 Gy irradiated BM-MSPCs (+Rx; hatched blue bars) immediately after thawing (off-the-shelf use; dark grey area) or after a 3-day rescue culture (light grey area) showed no significant difference at the three ratios as indicated (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Grey bar shows mean ± SD of unstimulated pooled T-cell proliferation. One representative experiment out of two is shown. b Representative histologic analysis of ectopic ossicles derived from native (non-irradiated, upper pictures) and irradiated (Rx; 30 Gy, lower pictures) BM-MSPC (n = 6 per group) 6 weeks after subcutaneous transplantation into immunocompromized NSG recipient mice. Bone formation via a vascularized cartilage intermediate was evident in hematoylin and eosin (HE; left panels) as well as Movat’s pentachrome (Movat; middle panels) staining. Vimentin staining (right panels) indicating persistence of reticular stromal cells (MSPCs) within the ectopic ossicles which showed infiltration by (human (hu.) Vimentin-negative) murine hematopoiesis as described previously only for native (non-irradiated) BM-MSPCs [27]. Scale bars are 100 μm in main histophotographs and 1 mm in inserts (showing overview of a section through the entire ossicle; dotted rectangles indicate the regions from where the magnified main pictures were derived). n.s. Not significant, pPBMC Pooled peripheral mononuclear cell
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4666276&req=5

Fig4: Irradiated MSPC maintain their immunomodulatory potency in vitro and their differentiation capacity in vivo. a Direct comparison of the inhibition of phytohemagglutinin (PHA)-induced T-cell proliferation (green bar) by non-irradiated bone marrow (BM)-MSPCs (-Rx; blue bars) versus 30 Gy irradiated BM-MSPCs (+Rx; hatched blue bars) immediately after thawing (off-the-shelf use; dark grey area) or after a 3-day rescue culture (light grey area) showed no significant difference at the three ratios as indicated (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Grey bar shows mean ± SD of unstimulated pooled T-cell proliferation. One representative experiment out of two is shown. b Representative histologic analysis of ectopic ossicles derived from native (non-irradiated, upper pictures) and irradiated (Rx; 30 Gy, lower pictures) BM-MSPC (n = 6 per group) 6 weeks after subcutaneous transplantation into immunocompromized NSG recipient mice. Bone formation via a vascularized cartilage intermediate was evident in hematoylin and eosin (HE; left panels) as well as Movat’s pentachrome (Movat; middle panels) staining. Vimentin staining (right panels) indicating persistence of reticular stromal cells (MSPCs) within the ectopic ossicles which showed infiltration by (human (hu.) Vimentin-negative) murine hematopoiesis as described previously only for native (non-irradiated) BM-MSPCs [27]. Scale bars are 100 μm in main histophotographs and 1 mm in inserts (showing overview of a section through the entire ossicle; dotted rectangles indicate the regions from where the magnified main pictures were derived). n.s. Not significant, pPBMC Pooled peripheral mononuclear cell

Mentions: To address the question whether the T-cell inhibitory function of MSPCs in this assay format is cell contact-dependent we performed additional experiments directly comparing randomly selected MSPCs from BM, WAT and UC in parallel either in direct cell–cell contact with the pooled PBMCs or in transwell cultures separating MSPCs (in the lower compartment) from PBMCs (in the transwell insert). Results revealed significant inhibition of PHA-induced T-cell mitogenesis independent of cell–cell contact. Inhibition of T-cell mitogenesis was significantly more efficient at PBMC:MSPC ratios of 1:1 and 1:10 at direct cell–cell contact. The allogeneic MLR resulting from cross-stimulation of the pooled PBMCs in the same assay format at day 7 was more significantly inhibited by UC-MSPCs at most PBMC:MSPC ratios. Inhibition of the MLR by BM- and UC-MSPCs was less efficient thus in part resembling data in Fig. 3 and published results. Also in the allogeneic MLR situation, depending on the PBMC:MSPC ratio, a cell–cell contact-independent inhibition of T-cell proliferation was observed (Additional file 5: Figure S4). Whether the simple standard assay testing MSPCs in direct contact with PBMCs, rather than the more complex transwell assay, might be able to predict the therapeutic MSPC potency in vivo needs to be determined in prospective clinical trials. Furthermore, we evaluated if 30 Gy gamma-irradiation of freshly thawed or cultured BM-MSPCs can be introduced as a putative safety measure before cells will be applied in vivo. Our results showed that irradiation did not influence their immunosuppressive potency (Fig. 4a). Interestingly, 30 Gy irradiation did not affect the differentiation potential of BM-MSPCs. We analyzed cartilage and bone formation of irradiated and non-irradiated BM-MSPCs in NSG mice and found that despite irradiation human Vimentin+ BM-MSPCs survived for up to 12 weeks in the immunocompromized animals and maintained their potential to form bone via a vascularized cartilage intermediate as recently described [27]. Hematoxylin and eosin as well as Movat’s pentachrome staining clearly demonstrated hypertrophic cartilage, osteoid and mineralized bone formation accompanied by immigration of murine marrow (Fig. 4b). This outcome extends a recent observation by Bianco and coworkers [30] showing that cartilage constructs generated from human BM-MSPCs ex vivo maintain their bone formation potential even when irradiated before transplantation into immunodeficient mice. These authors elegantly demonstrated that cartilage differentiation of BM-MSPCs in vitro is reversible and can be reverted, despite irradiation, in vivo, resulting in the generation of stromal hematopoietic niche-forming cells [30]. The goal of our experiments, in this study, was to determine whether BM-MSPCs are still capable of initiating patent chondrogenesis and subsequent osteogenesis after irradiation in advance of differentiation. Our observation that 30 Gy irradiation of human BM-MSPCs ex vivo did not impair cartilage and bone formation in vivo does not exclude the possibility that Vimentin+ stromal niche elements observed particularly in areas of hematopoiesis immigration could be derived from intermediate chondrocytes. A note of caution thus needs to be drawn regarding applicability of irradiated BM-MSPCs which might maintain their differentiation capacity if applied solely for immunomodulatory purposes. Whether unintended differentiation (as shown in this study and others [30, 31]) has to be considered a firm risk after systemic application needs to be assessed separately. Another relevant consequence of ex vivo irradiation may be premature MSPC senescence resulting in impaired immunomodulatory efficiency in vivo [32].Fig. 4


A robust potency assay highlights significant donor variation of human mesenchymal stem/progenitor cell immune modulatory capacity and extended radio-resistance.

Ketterl N, Brachtl G, Schuh C, Bieback K, Schallmoser K, Reinisch A, Strunk D - Stem Cell Res Ther (2015)

Irradiated MSPC maintain their immunomodulatory potency in vitro and their differentiation capacity in vivo. a Direct comparison of the inhibition of phytohemagglutinin (PHA)-induced T-cell proliferation (green bar) by non-irradiated bone marrow (BM)-MSPCs (-Rx; blue bars) versus 30 Gy irradiated BM-MSPCs (+Rx; hatched blue bars) immediately after thawing (off-the-shelf use; dark grey area) or after a 3-day rescue culture (light grey area) showed no significant difference at the three ratios as indicated (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Grey bar shows mean ± SD of unstimulated pooled T-cell proliferation. One representative experiment out of two is shown. b Representative histologic analysis of ectopic ossicles derived from native (non-irradiated, upper pictures) and irradiated (Rx; 30 Gy, lower pictures) BM-MSPC (n = 6 per group) 6 weeks after subcutaneous transplantation into immunocompromized NSG recipient mice. Bone formation via a vascularized cartilage intermediate was evident in hematoylin and eosin (HE; left panels) as well as Movat’s pentachrome (Movat; middle panels) staining. Vimentin staining (right panels) indicating persistence of reticular stromal cells (MSPCs) within the ectopic ossicles which showed infiltration by (human (hu.) Vimentin-negative) murine hematopoiesis as described previously only for native (non-irradiated) BM-MSPCs [27]. Scale bars are 100 μm in main histophotographs and 1 mm in inserts (showing overview of a section through the entire ossicle; dotted rectangles indicate the regions from where the magnified main pictures were derived). n.s. Not significant, pPBMC Pooled peripheral mononuclear cell
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666276&req=5

Fig4: Irradiated MSPC maintain their immunomodulatory potency in vitro and their differentiation capacity in vivo. a Direct comparison of the inhibition of phytohemagglutinin (PHA)-induced T-cell proliferation (green bar) by non-irradiated bone marrow (BM)-MSPCs (-Rx; blue bars) versus 30 Gy irradiated BM-MSPCs (+Rx; hatched blue bars) immediately after thawing (off-the-shelf use; dark grey area) or after a 3-day rescue culture (light grey area) showed no significant difference at the three ratios as indicated (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Grey bar shows mean ± SD of unstimulated pooled T-cell proliferation. One representative experiment out of two is shown. b Representative histologic analysis of ectopic ossicles derived from native (non-irradiated, upper pictures) and irradiated (Rx; 30 Gy, lower pictures) BM-MSPC (n = 6 per group) 6 weeks after subcutaneous transplantation into immunocompromized NSG recipient mice. Bone formation via a vascularized cartilage intermediate was evident in hematoylin and eosin (HE; left panels) as well as Movat’s pentachrome (Movat; middle panels) staining. Vimentin staining (right panels) indicating persistence of reticular stromal cells (MSPCs) within the ectopic ossicles which showed infiltration by (human (hu.) Vimentin-negative) murine hematopoiesis as described previously only for native (non-irradiated) BM-MSPCs [27]. Scale bars are 100 μm in main histophotographs and 1 mm in inserts (showing overview of a section through the entire ossicle; dotted rectangles indicate the regions from where the magnified main pictures were derived). n.s. Not significant, pPBMC Pooled peripheral mononuclear cell
Mentions: To address the question whether the T-cell inhibitory function of MSPCs in this assay format is cell contact-dependent we performed additional experiments directly comparing randomly selected MSPCs from BM, WAT and UC in parallel either in direct cell–cell contact with the pooled PBMCs or in transwell cultures separating MSPCs (in the lower compartment) from PBMCs (in the transwell insert). Results revealed significant inhibition of PHA-induced T-cell mitogenesis independent of cell–cell contact. Inhibition of T-cell mitogenesis was significantly more efficient at PBMC:MSPC ratios of 1:1 and 1:10 at direct cell–cell contact. The allogeneic MLR resulting from cross-stimulation of the pooled PBMCs in the same assay format at day 7 was more significantly inhibited by UC-MSPCs at most PBMC:MSPC ratios. Inhibition of the MLR by BM- and UC-MSPCs was less efficient thus in part resembling data in Fig. 3 and published results. Also in the allogeneic MLR situation, depending on the PBMC:MSPC ratio, a cell–cell contact-independent inhibition of T-cell proliferation was observed (Additional file 5: Figure S4). Whether the simple standard assay testing MSPCs in direct contact with PBMCs, rather than the more complex transwell assay, might be able to predict the therapeutic MSPC potency in vivo needs to be determined in prospective clinical trials. Furthermore, we evaluated if 30 Gy gamma-irradiation of freshly thawed or cultured BM-MSPCs can be introduced as a putative safety measure before cells will be applied in vivo. Our results showed that irradiation did not influence their immunosuppressive potency (Fig. 4a). Interestingly, 30 Gy irradiation did not affect the differentiation potential of BM-MSPCs. We analyzed cartilage and bone formation of irradiated and non-irradiated BM-MSPCs in NSG mice and found that despite irradiation human Vimentin+ BM-MSPCs survived for up to 12 weeks in the immunocompromized animals and maintained their potential to form bone via a vascularized cartilage intermediate as recently described [27]. Hematoxylin and eosin as well as Movat’s pentachrome staining clearly demonstrated hypertrophic cartilage, osteoid and mineralized bone formation accompanied by immigration of murine marrow (Fig. 4b). This outcome extends a recent observation by Bianco and coworkers [30] showing that cartilage constructs generated from human BM-MSPCs ex vivo maintain their bone formation potential even when irradiated before transplantation into immunodeficient mice. These authors elegantly demonstrated that cartilage differentiation of BM-MSPCs in vitro is reversible and can be reverted, despite irradiation, in vivo, resulting in the generation of stromal hematopoietic niche-forming cells [30]. The goal of our experiments, in this study, was to determine whether BM-MSPCs are still capable of initiating patent chondrogenesis and subsequent osteogenesis after irradiation in advance of differentiation. Our observation that 30 Gy irradiation of human BM-MSPCs ex vivo did not impair cartilage and bone formation in vivo does not exclude the possibility that Vimentin+ stromal niche elements observed particularly in areas of hematopoiesis immigration could be derived from intermediate chondrocytes. A note of caution thus needs to be drawn regarding applicability of irradiated BM-MSPCs which might maintain their differentiation capacity if applied solely for immunomodulatory purposes. Whether unintended differentiation (as shown in this study and others [30, 31]) has to be considered a firm risk after systemic application needs to be assessed separately. Another relevant consequence of ex vivo irradiation may be premature MSPC senescence resulting in impaired immunomodulatory efficiency in vivo [32].Fig. 4

Bottom Line: Reports on functional heterogeneity of MSPCs created additional uncertainty regarding donor and organ/source selection.Comparing various MSPCs revealed significant potency inconsistency and generally diminished allo-immunosuppression compared to dose-dependent inhibition of mitogenesis.Gamma-irradiation to block unintended MSPC proliferation did not prohibit chondrogenesis and osteogenesis in vivo, indicating the need for alternative safety strategies.

View Article: PubMed Central - PubMed

Affiliation: Experimental and Clinical Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center, Paracelsus Medical University, Salzburg, Austria. nina.ketterl@pmu.ac.at.

ABSTRACT
The inherent immunomodulatory capacity of mesenchymal stem/progenitor cells (MSPCs) encouraged initiation of multiple clinical trials. Release criteria for therapeutic MSPCs cover identity, purity and safety but appropriate potency assessment is often missing. Reports on functional heterogeneity of MSPCs created additional uncertainty regarding donor and organ/source selection. We established a robust immunomodulation potency assay based on pooling responder leukocytes to minimize individual immune response variability. Comparing various MSPCs revealed significant potency inconsistency and generally diminished allo-immunosuppression compared to dose-dependent inhibition of mitogenesis. Gamma-irradiation to block unintended MSPC proliferation did not prohibit chondrogenesis and osteogenesis in vivo, indicating the need for alternative safety strategies.

Show MeSH
Related in: MedlinePlus