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

Sara Bucar,Ana Fernandes-Platzgummer,João Coutinho Milhano,Íris Caramalho,André Dargen De Matos Branco,Cláudia L Da Silva,Márcia F Mata,Joaquim M S Cabral

doi:10.1186/s13287-021-02474-8

Abstract

BackgroundUmbilical 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.MethodsUCB 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.ResultsMSC 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.ConclusionsAT 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.

Highlights

Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/ progenitor cells (HSPC)
Despite human platelet lysate (HPL) being considered an alternative to fetal bovine serum (FBS) for clinical-scale manufacturing of mesenchymal stromal cells (MSC), further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells
In order to establish MSCbased feeder layer (FL) under xenogeneic-free (XF) conditions, i.e., MSC expanded without FBS-containing medium, half of the cells were subjected to two adaptive passages with low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% (v/v) fibrinogen-depleted human platelet lysate (HPL) (UltraGROTM-PURE; kindly provided by AventaCell Biomedical Corp., United States of America (USA)) (Certificate of Analysis (CoA) provided), while the other half continued to be expanded in low glucose DMEM with 10% MSC-qualified FBS (both supplemented with 1% (v/v) AntibioticAntimycotic (A/A) (Gibco, USA))

Summary

Introduction

Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/ progenitor cells (HSPC). Despite being a readily available source, with lower immunogenicity and lower risk of development of graft-vs-host disease (GVHD) compared to other sources, the low cell dose in a single UCB unit constitutes a major limitation [3] In this context, the majority of HCT with UCB cells were initially limited to children weighing 20–40 kg [4]. Several protocols were developed to promote ex vivo expansion of UCB HSPC, including the use of different media, cytokines, growth factors, and more recently the use of small molecules and chemical compounds [6,7,8] Alongside these approaches, BM mesenchymal stromal cells (MSC) have been used in a co-culture system to support the ex vivo expansion and maintenance of HSPC. Infusion of MSC proved to be safe, was associated with decreased incidence of acute GVHD, and reduced transplant-related mortality [14, 15]

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Conclusion