Laboratory Synthesis of Viable Primary Islet Beta Cells

This report delves into the laboratory synthesis of viable primary islet beta cells, a promising approach for treating diseases like diabetes. The study explores the in vitro generation of functional human pancreatic beta cells using human embryonic and induced pluripotent stem cells. The research, conducted by Pagliuca and colleagues, details the methods used to differentiate stem cells into glucose-responsive beta cells that mimic the characteristics of human islet beta cells. The report highlights the challenges in producing these cells, such as the presence of poly-hormonal cells, and the techniques used to overcome them, including transcriptional analysis, calcium signaling, and the introduction of retinoic acid and hedgehog inhibitors. The findings demonstrate that the synthesized beta cells secrete insulin in response to glucose, exhibit ultrastructural similarities to human islet cells, and effectively treat hyperglycemia in mice, suggesting a potential cure for diabetes through cell transplantation. This research is a breakthrough in the field of stem cell research and regenerative medicine.

Running head: LABORATORY SYNTHENSIS OF VIABLE PRIMARY ISLET Β CELLS 1
Laboratory synthesis of Viable Primary Islet β cells
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LABORATORY SYNTHESIS OF VIABLE PRIMARY ISLET Β CELLS 2
Laboratory Synthesis of Viable Primary Islet β cells
Some human diseases remain with cell replacement as the most effective therapy since
the already available treatments for example chemotherapy have proved ineffective. For example
diabetes, a condition that is rapidly affecting many people of across all the socio-economic status
can best be treated through tissues transplantation which is derived from the human stem cells.
Type 1 diabetes occurs as a result of an immune response that leads to the self-destruction of the
Beta cells (β) of the pancreas. Type 2 diabetes occurs when the peripheral tissues become insulin
resistant or when the pancreatic β cells experience dysfunction. The failure of these cells to
secrete insulin hormone leads to the unchecked irregular levels of glucose in the body which
have detrimental impacts on the body physiology. A ray of hope for the treatment of these
diseases was the discovery of human pluripotent stem cells (hPSC) that facilitate the in vitro
generation of cells even those that were could prove challenging to produce like the neurons
(Shiba et al., 2012). Production of such cells in the laboratory is vital for it provided cells for
research in drug discovery and cell transplantation. However, the hPSC derived β cells (SC-β)
are insufficient and fall short of many physiological characteristics of a bona fide β cell.
Therefore, the steady uniform supply of SC-β is required to suffice the high amount of these cells
needed for the pharmaceutical companies to research on the development of new drugs that
enhance the functioning of pancreatic β cells. Type 1 diabetic patients treated with cadaveric
human cells through transplantation are capable of producing their insulin independently after
five years or longer than this. So, in vitro massive production of functional human β cells could
check the highlighted challenges and even provide genetically diverse cells for sufficient for
research and offer the cure to many millions of people across the world. Having noted the
problems mentioned above, Pagliuca and colleagues embarked on research that would facilitate

LABORATORY SYNTHESIS OF VIABLE PRIMARY ISLET Β CELLS 3
in vitro production of functional pancreatic β cells (Pagliuca et al., 2014). The outcomes of this
research were that SC- β cells could secrete insulin when subjected to varying amounts of
glucose, the SC- β cells produced to resemble the human islet β cells regarding their
ultrastructure and expression of genes and that these cells check hyperglycemia when introduced
to diabetic mice. This article will discuss the findings as mentioned earlier in the study as well as
highlight the methods employed in the research.
Pagliuca and colleagues obtained human embryonic stem cells (hESC) and human-
induced pluripotent stem cells (hiPSC) and reproduced them using a scalable suspension-based
culture system which they later differentiated into glucose responsive β cells. The pancreatic
progenitor cells have an in vitro ability to regenerate into β cells that secrete can not only secrete
insulin but also form others with abnormal phenotypes secreting glucagon hormone. Even, these
co-reproduced pancreatic cells cannot express a bona fide β cell marker, for example, NKX6-1
and PDX1 (Narayanan et al., 2013). The abnormal cells (poly-hormonal) are do not respond to
glucose simulations, the task here is only to regenerate those cells that respond to glucose
challenge just like the human islets. Noting that the abnormal pancreatic β cells resemble human
fetal cells as opposed to the insulin-secreting cells that resemble the adult β cells (Hrvatin et al.,
2014), a transcriptional analysis was carried out to distinguish the two cell categories. First, the
progenitor cell culture for production of high insulin-producing cells was obtained by
introducing low concertation of retinoic acid and hedgehog inhibitors into the literature for an
extended duration to ensure the poly-hormonal cells were eliminated. Using sequentially
introduced levels of glucose only SC- β cells obtained from hESC and hiPSC exhibited glucose-
stimulated insulin secretion property and depolarization by potassium chloride solution (KCl). In

LABORATORY SYNTHESIS OF VIABLE PRIMARY ISLET Β CELLS 4
contrast, the hPSC cells produced small amounts of insulin upon subjection to sequentially added
amounts of glucose and did not well respond to depolarization test.
Secondly, the number of calcium ions influx in the cells upon subjection to different
glucose levels was used to differentiate stem cells that could effectively secrete insulin from the
ineffective ones. This technique is called calcium signaling whereby a high amount of glucose
increase membrane permeability letting in calcium ions which induce insulin production
Mohammed et al., 20009). Using fluorescent microscopy calcium comparison of calcium influx
at both single cell and cluster basis was made possible. Calcium influx for SC-β was better than
the poly-hormonal cells and similar to primary human islet cells. Also, the stem cell-derived β
cells positively responded to cytoplasmic C-peptide and the nuclear protein (NKX6-1) staining
like the islet cells as opposed to hSPC which do not co-express the C-peptide protein. The steps
as mentioned above successive lead to the regeneration of functional (mono-hormonal)
pancreatic cells in vitro through the elimination of the insufficiently insulin-secreting poly-
hormonal cells.
From the above experiments, it can be ascertained that the in vitro SC-β regenerated cells
are in all ways physiologically similar to the human islet cells (Bruin et al., 2014). Furthermore,
the ultrastructural feature of packaging insulin protein into secretory granules of the in vitro
produced beta stem cells is mirrored in the adult human beta cells. The packaged insulin appears
pale surrounded by a light halo which condenses to form dark polygonal crystalline cores
encircled by a halo (Wellmann, Volk & Brancato, 1971). Both the mature and developing SC-β
granules were observed and proved to be similar to primary human beta cells without forming α-
like pellets that have been reported before in other studies (D'Amour et al., 2006). These cells
were later transplanted into an immune-challenged mouse and after two weeks increased human

LABORATORY SYNTHESIS OF VIABLE PRIMARY ISLET Β CELLS 5
insulin amounts were found in the bloodstream just like the human islet cells could have elicited
the response in the mice (Kroon et al., 2008). Moreover, it was noted that mice injected with
glucose quickly cleared it when they were injected with SC-β cells as opposed to the hPSC cells.
Such findings provide evidence that SC-β can treat hyperglycemia.
In conclusion, the in vitro synthesis of stem cells is a breakthrough for various disciplines
as it enhances the generation of potent human cells from human pluripotent stem cells.
Generation of function SC-β ameliorates many of the challenges that result from the use of poly-
hormonal cells.

LABORATORY SYNTHESIS OF VIABLE PRIMARY ISLET Β CELLS 6
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