Influence of islet purity on the proportion of smaller islets and graft outcomes in clinical transplantation

Abstract

The outcome of clinical islet transplantation has been influenced by various factors, including the volume of islet transplanted (IEQ), frequency of transplantation, islet size, and purity. Most studies have compared the outcomes with these factors individually. This review attempts to comprehend the key factors that influence the outcome of clinical islet transplantation by understanding and analyzing the relationship between them. The purity of the islet preparation is often overlooked, as it is often believed to have a higher level of purity associated with better clinical outcome. However, a recent study documented long-term outcome of transplantation is better when purity less than 50%. Various studies have documented the decrease in the proportion of small islets with an increase in purity. Poor clinical outcomes have been documented when the transplanted islet preparation contains a lower proportion of small islets. This is not only because small islets survive better with diffusion, but also the higher proportion of large islets is prone to producing overestimation in IEQ calculation, leading to inadequate transplantation volume. Vigorous purification processes may remove the potential stem cells or progenitor cells present in pancreas and lack the paracrine effect that supports the viability and function of transplanted islets.

Key Words: Islets of Langerhans transplantation; Purification; Islet size; Engraftment; Insulin independence

Core Tip: The interaction between islet purity and size distribution influences the results of islet transplantation in addition to islet volume. Highly purified preparation prevents the adverse reaction, but it also decreases the small islets proportion and supportive progenitor cells from the preparation. Reduction in small islets leading to overestimation of the islet transplanted, reduced graft viability and function. Smaller islets demonstrate superior survival, revascularization, and insulin secretion, since it survives better with diffusion when compared with larger islets. Hence, moderate rather than maximal purification may preserve small islets, paracrine support, and regenerative potential, improving long-term engraftment and insulin independence.

INTRODUCTION

Islet transplantation, a promising treatment for type 1 diabetes, has yet to fully achieve its potential. In the late 1960s, Lacy et al[1] successfully performed islet transplantation in a chemically induced diabetic rodent model[1,2]. Series of further enhancement in the human islet isolation, purification and administration of immunosuppressive agents had enabled us to proceed with human transplantation in 1980s, with limited success rate[3-5]. Introduction of corticosteroid-free immunosuppressive regimen in Edmonton protocol enhances the transplant outcome with up to 80% of insulin independency at the end of one-year follow-up[6,7]. Still long-term success rate of islet transplantation doesn’t quite meet the mark. Following transplantation, less than 50% of the individual remains insulin-independent at the end of a five-year follow-up period[8,9].

Several factors have been reported to influence the outcome of islet transplantation, including the volume of islet transplanted (IEQ) - islet equivalent, frequency of transplantation, islet size, and purity[8,10-12]. One islet equivalent is defined as the volume of islet corresponding to 150 µm in diameter[13,14]. This unit serves as the gold standard for measuring the transplanted volume, irrespective of the islet’s actual size. Studies have demonstrated that the volume of IEQ is decisive for successful transplantation, with a minimum requirement of at least 3500-10000 IEQ/kg[6-8]. Islet size also impacts its viability and function post-isolation during in vitro culture and post-transplantation. Smaller islet sizes (< 125 µm) tend to thrive better due to their diffability and thus maintaining higher insulin-secreting capacity[10,12,15]. In vivo studies have demonstrated a higher Insulin secreting capacity reflected by a higher stimulation index, in smaller pancreatic islets[15-17].

In the early periods of islet transplantation, severe inflammatory reactions were observed due to the impure nature of the islet product[18-20]. Islet purity refers to the proportion of actual islets in the preparation in relation to the amount of non-islet cells like exocrine cells (Figure 1)[21]. Islet purification during islet allo-transplantation is unavoidable due to large tissue pellet at the end of pancreas digestion step. However, with the advent of the gradient purification technique, islet preparation has been purified with minimal exocrine acinar cells. Nevertheless, despite achieving high levels of purity, studies have consistently reported that highly purified islet preparations are associated with less favorable long-term outcomes compared to their less purified counterparts[11]. This may be attributed to the absence of the subtle paracrine dialogue between exocrine cells and islets, possibly a vital interplay lost in the pursuit of purity[11,18]. While most studies have assessed these factors separately, they are in fact closely interconnected and influence each other in meaningful ways. Consequently, the present review aimed to elucidate the impact of islet purity on the proportion of smaller islets and to discuss their influence on the IEQ calculation, viability, functionality, and clinical transplantation outcome.

Figure 1 Schematic diagram illustrating the alterations in the islet size proportion and other cellular composition during the purification process. This image was created with BioRender.

PURIFICATION OF THE ISOLATED ISLETS

In the early ventures of islet transplantation, the use of unpurified islets resulted in various adverse reactions, including an elevated risk of portal vein thrombosis, enhanced rejection, and increased islet damage[18,22-24]. However, these studies were predominantly conducted in rodents prior to the implementation of the Edmonton Protocol. The transplantation sites utilized in these studies were predominantly the renal capsule, which is not typically the preferred site for clinical islet transplantation[18,25]. Subsequent studies in human subjects, conducted under the framework of the Edmonton Protocol, revealed a somewhat paradoxical yet compelling insight: Islet preparations with purity levels below 50% were associated with more favorable long-term clinical outcomes than their highly purified counterparts with purity greater than 50%[11]. This observation introduced a subtle complexity to the evolving narrative of islet transplantation, where, intriguingly, a lesser degree of purity at times yielded greater clinical benefit[11].

Now-a-days purification of islets were performed using density gradient centrifugation in a specialized cell processing device, most commonly in COBE 2991 cell processor[26]. During the density gradient centrifugation, lighter islets (density approximately 1.070 g/cm³) migrate to the top “pure” layer (interface), while denser acinar/exocrine tissue pellets at the bottom[27,28]. Islet purification process removes lots of single cells (ex: Ductal cells, progenitor cells, endothelial cells, etc.). Notably, ductal cells are considered potential precursors of islet cells, with the capacity to transdifferentiate into alpha cells and, subsequently, into beta cells following transplantation[11]. Yang et al[29] demonstrated a higher expression of stem cell markers, such as cytokeratin-19 and pancreatic and duodenal homeobox- 1 mRNA, in the unpurified preparation (purity of 43.6% ± 6.29%) when compared to moderately purified (65.3% ± 4.40%) and highly purified (77.6% ± 6.36%) islet preparations[29]. Nevertheless, stem cells remained detectable even within highly purified islet preparations[29,30].

In laboratory settings, adherence to rigorous purification protocols often results in reduced islet yield. Purification entails the loss of islets, as evidenced by studies showing that only 30% to 50% of the isolated islets were successfully transplanted post-purification[31,32]. Consequently, the stringent purification protocol leads to an increase in the number of failed isolations, necessitating the use of two or more pancreases to perform single islet transplantation with adequate volume[31]. Furthermore, highly purified preparations are devoid of non-islet pancreatic cell populations, such as acinar, ductal, endothelial, and mesenchymal stem cells[11]. These cells are recognized for their contribution to islet function and survival through paracrine signaling. The intra-islet capillary endothelial cells and beta cells are the sources of various trophic factors, including vascular endothelial growth factor A, hepatocyte growth facto, endothelin-1, angiopoietin 1, basement membrane proteins and Insulin, which are known to maintain the survival and functionality of both beta cells and endothelial cells[33-35]. The absence of this supportive microenvironment may further compromise islet viability and insulin secretion capacity, underscoring a potential biological trade-off between islet purity and overall transplant efficacy.

CHANGES IN PROPORTION OF SMALL ISLETS IN THE PROCESS OF PURIFICATION

Studies reported on the effect of purification on the islet size distribution was limited in the literature. Notably, a multi-center analysis conducted by Balamurugan et al[36] reported consolidated data of 1017 cases (in three different era of islet transplantation) in which purity of the islet purity increases over the period of time associated with significant increase in IEQ/islet particle ratio. IEQ/islet particle ratio is used to estimate the average size of islets in a preparation; higher the values denote the increase in larger islet composition. Paushter et al[37] reported 4% to 5% reduction in small islets (50 µm to 150 µm) contribution to IEQ post-purification with compensatory rise in large islet (> 150 µm). These finding clearly demonstrates the preferential loss of small islets during the process of high purification. Unpublished retrospective data (n = 25) from our lab shows the similar trends. In the pre-purification stage, smaller islets (50 µm to 200 µm) comprised 42.18% ± 9.92% of the total IEQ. Highly purified islet preparations resulted in a notable decrease in the contribution of smaller islets to the total IEQ, reducing to 33.55% ± 13.21% (P = 0.003). Conversely, there was a proportionate increase in the contribution of larger islets (> 200 µm) to the total IEQ, reaching 66.45% ± 13.21% (P = 0.003). In contrast, medium and low purity levels nearly maintained the contribution of smaller islets to the IEQ at 44.43% ± 11.83% (P = 0.34), similar to the pre-purification stage. Therefore, there is a 10.88% (P < 0.001) reduction in the contribution of smaller islets to the total islet volume during the medium to high purification stages, accompanied by a reciprocal increase in the contribution of larger islets. The cause of the loss of smaller islets remains poorly understood. It may be due to the overlap density of the smaller islets with exocrine acini.

This suggests a relative enrichment of larger islets during the purification process, as smaller islets are preferentially lost. However, this shift in size distribution is accompanied by a substantial reduction in functional and viability parameters. Specifically, the stimulation index decreased (from 3.3 ± 3.3 to 2.7 ± 2.7; P < 0.001) with increasing purity (from 61.8% ± 18.3% to 65.1% ± 16.4%; P < 0.01), and islet viability also exhibited a modest but statistically significant decline (P < 0.01)[36]. These findings suggest that larger islets, while more likely to be retained during purification, possess lower glucose responsiveness. This is likely attributed to limited oxygen and nutrient diffusion, resulting in core hypoxia[38].

THE SMALLER ISLETS BEAR A VITAL INFLUENCE PROFOUNDLY SHAPING THE CLINICAL OUTCOMES OF ISLET TRANSPLANTATION

Islets, like other endocrine organs, receive an abundant vascular supply (ten times greater than the pancreatic acinar cells) to meet their metabolic demands[39-41]. When isolated from native pancreatic parenchyma, islets devascularize and rely solely on diffusion for their nutrition (glucose and oxygen), hormone secretion, and excretion of metabolic waste[42]. Due to their smaller size and fewer number of cell groups, small islets exhibit superior survival rates through diffusion compared to large islets[16,17,43-45]. This leads to enhanced insulin-secreting capacity and improved clinical transplant outcomes. Studies have demonstrated that glycemic control and insulin independence post-transplantation are significantly better when the transplanted islet preparation contains a higher proportion of small islets compared to larger islets[12,15-17,45-47]. Furthermore, small islets have a higher cell density per IEQ. In an islet preparation containing small islets with a diameter of 50 µm, the cell count per IEQ is 2235[48]. Conversely, when the islet size exceeds 200 µm, the cell count per IEQ is reduced to 824 cells[48]. The vascular network of a large islet is approximately doubled in size compared to a small islet, providing adequate supply of cells to the core[49]. Consequently, in terms of cell counts, viability, insulin secreting capacity and transplantation outcome small islets demonstrate a superior advantage[10].

IMPACT OF ISLET SIZE ON IEQ CALCULATION

The crucial factor influencing the islet transplantation outcome, as highlighted in the Edmonton protocol, is the quantity of islets transplanted (approximately 3500-10000 IEQ per kilogram)[50]. IEQ, the widely used measurement system for islet volume, mathematically converts the two-dimensional islet dimension measured under a microscope into three-dimensional volume using a conversion factor determined for each islet size bin of 50 µm increments[7,13]. Numerous studies have persistently challenged the IEQ calculation method, pointing to its flawed assumption that all islets are perfect spheres[49,51]. In truth, islets often take on an ellipsoidal form-especially the larger ones-bearing a circularity index that typically falls between 0.6 and 0.74[49,52,53]. This quiet geometric truth casts a shadow over the precision of standardized quantification[51]. The binning nature of IEQ calculation further contributes to the overestimation of up to 50%[54,55].

To elucidate the extent of overestimation of the IEQ in relation to the actual size of the islets, we conducted a serial section histomorphometric study to measure the actual volume and the volume calculated using the IEQ based on the diameter of the islets[53]. The IEQ calculated using diameter as used in clinical islet transplantation was found to be 87.51% overestimated compared to the actual volume measured using serial sections. Notably, when the islet size is less than 125 µm, the overestimation is 59.76%. Conversely, in the case of large islets, the overestimation reaches 111.7%[53]. The presence of a consistent level of overestimation across various islet sizes is inconsequential and may be neglected as a fixed variation between two measuring units. However, it is crucial to acknowledge that the overestimation significantly increases with the size of the islet. Consequently, if the islet preparation contains a higher proportion of large islets, due to this error in IEQ calculation, the actual IEQ may not meet the minimum required quantity as stipulated by the Edmonton protocol. Several studies have reported adverse outcomes associated with an increased proportion of large islets (higher IEQ/islet particle number or islet index) in the transplanted preparation, although these findings have not been interpreted in terms of IEQ error. Therefore, purity-driven changes in the islet size proportion may potentially increase the risk of IEQ overestimation, leading to a reduction in the transplanted islet volume and consequently, poor outcomes (Figure 2).

Figure 2 Flowchart describing the various mechanism by which the highly purified islet preparation providing poor transplant outcome in comparison to the medium or low purified preparation. IEQ: Islet transplanted.

LIMITATIONS OF LOW-PURITY ISLET TRANSPLANTATION

The potential advantages of preserving the small islets by transplanting the lower purity islet preparation may be tempered by several disadvantages associated with low-purity islet preparations. Low purity preparation often comes with higher pellet volume which is associated with increased portal pressure and thrombosis[56]. Degradation of acinar cells from low purity preparation may trigger the instant blood-mediated inflammatory reaction and peri-transplant inflammation, which account for immediate post transplantation loss of islets[57,58]. Thus optimal cutoff of purification need to be determined to balance the benefit of preserving the smaller islets and minimizing the adverse outcome related to the low purity preparation.

FUTURE DIRECTIONS

Developing optimal purification method to preserve the small islets and stem cells is required to improve the islets transplant outcome. Further clinical studies required with variable purity and constant islet index to validate the outcome influenced by the purity and islet dimension.

CONCLUSION

Islet purity stands as a double-edged sword in clinical transplantation-while essential for minimizing immunogenic risk, excessive purification may strip away vital cellular components such as ductal progenitors and smaller islets, undermining viability, regenerative potential, and paracrine support. A balanced approach, rather than maximal refinement, may thus be the key to achieve enduring graft function and favorable long-term outcomes. The need for newer islet purification techniques to recover smaller islets during the process will not only increase islet yield but also improve the favourable long-term transplant outcome. Developing techniques to transplant unpurified islets in alternative sites other than the portal vein will facilitate the transplantation of more islets containing ductal and progenitor cells.

ACKNOWLEDGEMENTS

We extend our deepest gratitude to the courageous families who generously donated their loved one’s organs and tissues for biomedical research. Such important research like this would not be possible without this selfless gift of hope. Thanks to Network for Hope (Louisville and Cincinnati) and Lifeline of Ohio, Columbus for supporting these special families and providing human research pancreases to Dr. Balamurugan’s islet lab. We also thank members of Dr. Balamurugan’s islet lab for their contributions to islet research and transplant and thanking funding agencies.