Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

Abstract

© Ferrata Storti Foundation IntroductionSince the first clinical trial of mesenchymal stromal cells (MSC) 9,10 Another important issue regarding the clinical application of MSCs is their culture under serum-free conditions. The majority of clinical studies have used MSCs that were expanded in media supplemented with fetal bovine serum (FBS). 1,[11][12][13][14][15] To avoid the risks associated with the use of FBS, 16 platelet lysate (PL) was proposed as a supplement to tissue culture media for MSCs. 17 Recently, several studies showed that MSCs that were expanded in PL exhibited the same efficacy as MSCs cultured in serum-containing media for the treatment of GvHD. 18-22To date, clinical studies have used MSCs that have been generated from several individual donors. Considering the aforementioned inter-donor heterogeneity and the need for a large number of "off-the-shelf" MSCs, the establishment of MSC banks appears to be an indispensable strategy for providing a continuous supply of MSCs with predictable potency. To our knowledge, there are few established MSC banks worldwide, and these MSC banks were generated by separately isolating, expanding, and freezing MSCs from up to 10 donors in FBS-containing media. [23][24][25][26] In the current study, we report for the first time the establishment of a serum-free and GMP-compliant MSC bank generated from pooled bone marrow mononuclear cells (BM-MNCs) of multiple donors as a novel strategy to circumvent donor-to-donor variability. Clinical-grade MSC endproducts (MEPs) derived from the MSC bank were thoroughly assessed for their proliferation, differentiation, and, in particular, for the allosuppressive potential in vitro. Importantly, 81 MEPs were administered as a rescue therapy to 26 pediatric patients with severe steroid-refractory aGvHD in seven transplantation centers. Safety and efficacy of MEPs was compared to MSCs generated from a single or several individual donors that have been used in the GvHDclinical studies reported thus far. Methods Generation of MSC bank and clinical-grade MEPsBone marrow was collected from 8 healthy volunteers (age 21-45 years old) after written informed consent and after the approval of the local Ethics Committee (n. 275/09). BM-MNCs were enriched from the bone marrow aspirate by using the Sepax II NeatCell process (Biosafe, Eysins, Switzerland) and frozen individually. After thawing and washing these BM-MNCs were pooled. This pool of BM-MNCs from 8 donors was used to generate MSCs over 14 days in culture. After their detachment, passage 1 mesenchymal stromal cells (MSC-P1) were washed and aliquoted into 209 cryovials (each containing 1.5x10 6 MSC-P1). Cryopreserved vials with MSC-P1 were referred to as the MSC bank.To generate clinical-grade MEPs, MSC-P1 aliquots from the MSC bank were thawed and after washing they were expanded in medium containing 10% PL till the end of passage 2. These MSCs were re-suspended in cryomedium (0.9% NaCl containing 5% HSA and 10% DMSO), distributed in cryobags (each containing 1-3x10 6 MS...

Evaluation of the Secretome Profile and Functional Characteristics of Human Bone Marrow Mesenchymal Stromal Cells-Derived Conditioned Medium Suggest Potential for Skin Rejuvenation

“…Also, treatment with CM or 10× CM led to considerable increase in synthesis of basal levels of extracellular matrix component like elastin which is a key protein responsible for skin tightening and retention of skin elasticity. In a study published earlier, it has been documented that CM from adipose tissue has a similar effect on dermal fibroblast [17]. In addition, our BMMSC CM was able to restore the viability and ECM integrity following the induction of oxidative stress and UV-B damage.…”

“…The rationale for using a three donor pool of BMMSC as opposed to a single donor derived cells relates to the fact that MSCs including BMMSC from different donors show significant variability in their proteomic signature which may eventually be manifested in the composition of the CM [17] [18] [19]; and one of the ways to minimize this problem would be to use a pooled population of MSCs for CM production. In this communication, we report that this medium is highly enriched in factors that are known to stimulate cell proliferation and migration, angiogenesis, remodeling of ECM and re-epithelialization, and a combination of such diverse macro-and small molecules may inversely modulate the skin aging process.…”

Clinical Use of Mesenchymal Stromal Cells in the Treatment of Acute Graft-versus-Host Disease

“…Generated MSCs were frozen in 209 vials and labeled as an MSC bank. This novel manufacturing protocol is characterized by high potency and near-identity of individual doses, termed MSC-Frankfurt am Main (MSC-FFM) [24]. Recently, Bader et al [5] reported outcomes of 69 adult and pediatric patients who received a total of 212 doses of MSC-FFM for therapy-refractory aGvHD in a routine clinical setting in 23 allogeneic transplant centers from 6 countries.…”

“…Certain factors, including skin involvement and lower aGvHD grade, yielded a higher response rate to MSC application [25]. Possibly, inconsistent results of clinical studies are attributable to different or inconsistent pharmacological quality of MSCs resulting from the lack of a standardized methodology for MSC generation, dosing, and interdonor heterogeneity [5, 24]. Kuçi et al [24] developed a novel approach for MSC generation from pooled bone marrow mononuclear cells of 8 allogeneic, third-party donors to overcome donor-to-donor variability.…”

In Vivo Exposure to Inorganic Arsenic Alters Differentiation-Specific Gene Expression of Adipose-Derived Mesenchymal Stem/Stromal Cells in C57BL/6J Mouse Model

“…Additionally, our choice to pool samples may reflect a more clinically-relevant application of MSCs. For example, it has been proposed that pooled donor samples may be advantageous in overcoming the heterogeneity associated with the efficacy of MSC-based therapies (Kuçi et al, 2016). Thus, while we lose individual variance from pooling cells from different genetically identical donors, we gain an understanding of how an at-risk population may be influenced by environmental contaminants.…”