Critical quality attributes for identifying mesenchymal stromal cells with immunomodulatory and angiogenic fitness

A method of determining angiogenic fitness of derived mesenchymal stromal cells (MSC) comprising:

• determining a sample critical quality attribute (CQA) profile, the sample CQA profile comprising:
• a) from the sample harvested and cultured:
• a. in the presence of proinflammatory cytokines stimulation post-harvest:
• the mRNA levels of the following genes:
• SMAD7, HGF, ANG, IDO1, TSG101, CXCL8 and the level of VEGF protein;
• and/or
• b. in the absence of the proinflammatory cytokines stimulation post-harvest:
• the mRNA levels of the following genes:
• SOX9, TNFAIP6 and the level of VEGF protein;
• determining the level of similarity of said sample CQA profile to one or more control profiles, wherein (i) a high level of similarity of the sample CQA profile to an angiogenic specific control profile;
• (ii) a low level of similarity to a non-angiogenic specific control profile;
• and/or (iii) a higher level of similarity to an angiogenic specific control profile than to a non-angiogenic specific control profile indicates the cells have basal angiogenic fitness.

A method of determining quantitative values for a set of CQAs for assessing immunomodulatory fitness of samples of mesenchymal stromal cells (MSC), wherein the MSC samples are from donors and/or from samples grown using different critical processing parameters (CPPs), the method comprising:

• a) determining the values of the set of CQAs for each sample;
• wherein the set of CQAs comprise:
• a. from the sample cultured and harvested:
• i. in the presence of proinflammatory cytokines stimulation post-harvest:
• the mRNA levels of the following genes:
• THBS1, CCN2, EDN1, ACTA2, PDCD1LG2, TNFAIP6, ANGPT1, CXCL8;
• and/or
• ii. in the absence of the proinflammatory cytokines stimulation post-harvest:
• the mRNA levels of the following genes:
• CCN2, TSG101, THBS1, PDGFA, VEGFA, EDIL3, ACTA2, ANGPT1, ANG and the level of TGF-beta protein;
• and/or
• b. from a single cell suspension of the sample:
• cell diameter and cell circularity post-harvest without pro-inflammatory cytokine stimulation;
• b) testing each sample of MSC for immunomodulatory fitness using in vitro assays or obtaining immunomodulatory fitness status of the sample;
• c) conducting a regression analysis between each CQA of a) with the immunomodulatory fitness of b);
• determining minimum and maximum data values of each CQA of the samples and using multivariate dimension reduction statistical or mathematical methods to assign minimization or maximization functions to indicate whether higher or lower values are desirable based on the directionality of correlation respectively.

A method of determining quantitative values for a set of CQAs for assessing angiogenic fitness of samples of mesenchymal stromal cells (MSC), wherein the MSC samples are from different donors and/or from samples grown using different critical processing parameters (CPPs), the method comprising:

• a) determining the values of the set of CQAs for each sample;
• wherein the set of CQAs comprise:
• a. from the sample cultured and harvested:
• i. in the presence of proinflammatory cytokines post-harvest stimulation:
• the mRNA levels of the following genes:
• SMAD7, HGF, ANG, IDO1, TSG101, CXCL8 and the level of VEGF protein;
• and/or
• ii. in the absence of the proinflammatory cytokines post-harvest stimulation:
• the mRNA levels of the following genes:
• SOX9, TNFAIP6 and the level of VEGF protein;
• and
• b) testing each sample of MSCs for angiogenic fitness using in vitro assays or obtaining angiogenic fitness status of the sample;
• c) conducting a regression analysis between each sample CQA of a) with the angiogenic fitness of b);
• determining minimum and maximum data values of each CQA of the samples and using multivariate dimension reduction statistical or mathematical methods to assign minimization or maximization functions to indicate whether higher or lower values are desirable based on the directionality correlation respectively.