Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34+-enriched cells

Abstract

Background Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo. Methods UCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed. Results MSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells. Conclusions AT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.

Hypothermic Preservation of Adipose-Derived Mesenchymal Stromal Cells as a Viable Solution for the Storage and Distribution of Cell Therapy Products

“…The colony-forming unit (CFU) assay, which tested the myeloid differentiation potential of HSPC, was performed as part of the hematopoietic support assay. Cell culture without MSC FL typically originates an equal share of colony-forming unit granulocyte-macrophage (CFU-GM) and CFU-multilineage (CFU-Mix) for the expanded UCB cells, although the co-culture system increases the proportion of CFU-GM [ 27 , 30 ]. This difference was used to detect any loss of function by MSC(AT) during encapsulation.…”

“…However, HPL has also been associated with reduced MSC immunomodulation caused by an altered secretome and impaired inhibition of T- and NK-cell proliferation [ 46 , 47 ]. Additionally, the hematopoietic support capacity of MSC was demonstrated to be negatively affected by HPL supplementation as it was unable to retain certain hematopoietic stem and progenitor cell (HSPC) populations [ 30 ]. In our study, cells maintained in HPL-containing medium had slightly better encapsulation recovery and viability over time compared to FBS-based medium and comparable performance concerning the remaining assays.…”

“…This hematopoietic support ability of MSC can be quantified and used as an indicator for MSC potency or function. Our group has an extensive background with this co-culture expansion system and has contributed toward its translation potential [ 27 , 30 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. Our proposed hematopoietic support assay has the advantage of multiple quantifiable readouts (e.g., HSPC expansion fold change, metabolite quantification, HSPC immunophenotype, and percentages of colony-forming unit [CFU] populations) and an internal control (i.e., HSPC expansion only with exogenous cytokines).…”

Human umbilical cord blood mononuclear cells transplantation for perinatal brain injury

“…On the one hand, umbilical cord blood mesenchymal stem cells promote the proliferation of hematopoietic stem cells by secreting a variety of cytokines with hematopoietic support [24,25]; on the other hand, umbilical cord blood mesenchymal stem cells express adhesion molecules that interact with hematopoietic cells, regulate the adhesion between hematopoietic cells and stromal cells, and play an important role in hematopoietic cell implantation and homing [24]. Many experiments have shown that the combined transplantation of UCB-derived MSCs and HSCs can improve the success rate of hematopoietic stem cell transplantation [26][27][28]. Intracerebral transplantation of mesenchymal stem cells derived from human umbilical cord blood alleviated hypoxic-ischemic brain damage (HIBD) in rat neonates via migrating to damaged brain tissues to inhibit neuronal apoptosis [29].…”

Roles of mesenchymal stem cells and exosomes in interstitial cystitis/bladder pain syndrome

“…MSCs can be isolated from different adult tissues such as umbilical cord blood, bone marrow, adipose tissue, placenta, amniotic fluid, urine and other tissues. 7 , 8 , 9 , 10 , 11 They express low levels of major histocompatibility complex (MHC) class I molecules and MHC class II molecules, which enable them to have low immunogenicity. 12 In vitro, MSCs can differentiate into a range of cell types, including adipocytes, osteocytes, hepatocytes, chondrocytes, muscle, vascular smooth muscle cells and other connective tissues.…”