Stem cell bioengineering strategies to widen the therapeutic applications of haematopoietic stem/progenitor cells from umbilical cord blood

Abstract

Umbilical cord blood (UCB) transplantation has observed a significant increase in recent years, due to the unique features of UCB haematopoietic stem/progenitor cells (HSCs) for the treatment of blood-related disorders. However, the low cell numbers available per UCB unit significantly impairs the widespread use of this source for transplantation of adult patients, resulting in graft failure, delayed engraftment and delayed immune reconstitution. In order to overcome this issue, distinct approaches are now being considered in clinical trials, such as double-UCB transplantation, intrabone injection or ex vivo expansion. In this article the authors review the current state of the art, future trends and challenges on the ex vivo expansion of UCB HSCs, focusing on culture parameters affecting the yield and quality of the expanded HSC grafts: novel HSC selection schemes prior to cell culture, cytokine/growth factor cocktails, the impact of biochemical factors (e.g. O2 ) or the addition of supportive cells, e.g. mesenchymal stem/stromal cell (MSC)-based feeder layers) were addressed. Importantly, a critical challenge in cellular therapy is still the scalability, reproducibility and control of the expansion process, in order to meet the clinical requirements for therapeutic applications. Efficient design of bioreactor systems and operation modes are now the focus of many bioengineers, integrating the increasing 'know-how' on HSC biology and physiology, while complying with the GMP standards for the production of cellular products, i.e. through the use of commercially available, highly controlled, disposable technologies.

Human Adipose-Tissue Derived Stromal Cells in Combination with Hypoxia Effectively Support Ex Vivo Expansion of Cord Blood Haematopoietic Progenitors

“…Cord blood haematopoietic stem and progenitor cells (cbHSPCs) have attracted considerable interest as a full value alternative to bone marrow HSPCs. The number of cbHSPC transplants is increasing from year to year, but their low number in one cord blood sample is a significant limitation to expanded application of these cells [ 1 ]. In this connection, the development of ex vivo approaches for cbHSPC amplification and differentiation into certain haematopoietic lineages is a primary goal of cell technologies.…”

“…Until recently, most of such experiments were conducted on stromal and haematopoietic cells isolated from bone marrow [ 1 ]. The results of these studies demonstrated that MSCs, applied as a feeder layer, may considerably influence the co-cultured HSCs, in particular, changing the ratio of poorly differentiated and committed progenitors [ 3 , 4 , 6 , 7 ].…”

“…This resulted in the more pronounced formation of haematopoietic colonies and increased production of IL-6, IL-8 as compared to co-culture in standard conditions (20% O 2 ) [ 23 ]. In the case of hypoxia, improved expansion of CD34 + HSPCs in the presence of osteoblasts [ 24 ] and bone marrow [ 1 , 25 , 26 ] MSCs was demonstrated.…”

Developing a co-culture system for effective megakaryo/thrombopoiesis from umbilical cord blood hematopoietic stem/progenitor cells

“…Our results suggest the existence of inhibitory effects of FBS addition to the culture medium on the megakaryopoiesis ex vivo and provide insights on how a BM-MSC feeder layer, used as accessory cells, contribute either through direct cell-cell contact and/ or soluble factor signaling to modulate proliferation, maturation and development of Mks as well as platelet production from the expanded UCB progenitors. The protocol developed benefits from the use of UCB progenitors because these are readily available from simple collection procedures, having less risk of provoking graft-versus-host disease and a high proliferative rate in vitro compared with adult counterparts [35]. This study provides evidence that UCB CD34 þ -enriched cells differentiated in the presence of a BM-MSC feeder layer are able to generate polyploid Mks (DNA content >4N) and finally to produce functional platelets through the use of a serum-free system supplemented by cytokines (TPO and IL-3).…”

“…Overall, in our culture conditions, the differentiation potential of UCB CD34 þ cells, expanded by use of the Z9 cocktail, was shifted toward myeloid lineage, as previously reported [19], with significantly higher megakaryocytic content, considerably affecting the differentiation stage. Indeed, expanded cells in the Z9 condition Although FBS has been widely used as a culture medium supplement to support the growth and proliferation of several animal cells, the development of serum-free/animal originefree culture systems is crucial to meet the requirements of the regulatory agencies [34], improving the batch-to-batch consistency and safety of cellular products [35]. In this study, QBSF-60 was assessed as a medium for differentiation stage, supplemented by Cyt and/or Feeder conditions, resulting in a significantly higher EY, %CD41 and %CD42b and higher polyploid content (>4N, %) when compared with a nondefined medium containing FBS (IMDMþ10% FBS).…”