Glycoprotein Ibα Receptor Instability Is Associated with Loss of Quality in Platelets Produced in Culture

Abstract

The development of culture processes for hematopoietic progenitors could lead to the development of a complementary source of platelets for therapeutic purposes. However, functional characterization of culture-derived platelets remains limited, which raises some uncertainties about the quality of platelets produced in vitro. The aim of this study was to define the proportion of functional platelets produced in cord blood CD34+ cell cultures. Toward this, the morphological and functional properties of culture-derived platelet-like particles (PLPs) were critically compared to that of blood platelets. Flow cytometry combined with transmission electron microscopy analyses revealed that PLPs formed a more heterogeneous population of platelets at a different stage of maturation than blood platelets. The majority of PLPs harbored the fibrinogen receptor αIIbβ3, but a significant proportion failed to maintain glycoprotein (GP)Ibα surface expression, a component of the vWF receptor essential for platelet functions. Importantly, GPIbα extracellular expression correlated closely with platelet function, as the GPIIb+ GPIbα+ PLP subfraction responded normally to agonist stimulation as evidenced by α-granule release, adhesion, spreading, and aggregation. In contrast, the GPIIb+ GPIbα⁻ subfraction was unresponsive in most functional assays and appeared to be metabolically inactive. The present study confirms that functional platelets can be generated in cord blood CD34+ cell cultures, though these are highly susceptible to ectodomain shedding of receptors associated with loss of function. Optimization of culture conditions to prevent these deleterious effects and to homogenize PLPs is necessary to improve the quality and yields of culture-derived platelets before they can be recognized as a suitable complementary source for therapeutic purposes.

Microfluidic assessment of functional culture-derived platelets in human thrombi under flow

“…Platelets were defined by flow cytometry as events that had similar size (forward scatter) and surface (CD41 and CD42b) marker expression as blood platelets (Figure 3B, Supplementary Figure 2). While all CD42b + CDPs expressed CD41, only a fraction of CD41 + CDPs expressed CD42b as shown previously (Supplementary Figure 3) 12 . Using this protocol, we generated approximately 60 CD41 + CD42b + platelets and 30 CD41 + CD42b + megakaryocytes on Day 14 for every CD34 + cells seeded on Day 0 (Figure 3C).…”

“…These criteria ensured that larger cells, microparticles, and GPIIb + & GPIb − (CD41 + & CD42b − ) particles were excluded from our analysis. Omitting GPIIb + & GPIb − is critical as previous groups have shown that these platelet-like particles are not characteristic of functional platelets 12 .…”

“…Human peripheral blood CD34 + cells were differentiated into megakaryocytes and platelets using a 14 day protocol similar to a previously described method 12 . Progenitor cells were expanded for 4 days followed by differentiation in megakaryocyte conditions for an additional 10 days (Figure 3A).…”

“…Flow cytometry is the current gold standard for defining the yield and quality of CDPs based on their similarity in surface marker expression and scatter properties to blood platelets. Pre-activated platelets, megakaryocyte membrane particles and other non-functional platelet like particles (PLPs) may get classified as functional platelets by flow cytometry 1,12 . Some groups have demonstrated that CDPs can incorporate into mouse thrombi and respond to inhibitors of platelet function 2–4 .…”

Megakaryocyte polyploidization and proplatelet formation in low-attachment conditions

“…PLT components are shuttled and packaged along these extensions, termed proplatelets (proPLTs), before being sheared off by flowing blood [1]. MKs and their PLT progeny have been successfully derived in vitro from CD34 + hematopoietic stem and progenitor cells (HSPCs) from mobilized peripheral blood (mPB) [2, 3] and umbilical cord blood [4-8], as well as MK progenitor immortalized cell lines [9], induced pluripotent stem cells [10-12], and embryonic stem cells [13-15]. …”