Spatial profiling of scRNAseq signatures in human parathyroid glands

Awardee: Julie Ann Sosa

Institution: University of California at San Francisco

Grant Amount: $50,000

Funding Period: April 1, 2021 - March 31, 2022

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The objectives of this project were to: (1) utilize transcriptomic methods to define individual cell types within the human parathyroid, and (2) employ digital spatial profiling to visualize the localization of these cell types within the native parathyroid gland architecture. The developmental pilot phase work supported by the grant enabled us to establish a solid foundation of procedural optimization and proof of concept data for scaling our single cell sequencing efforts to a larger, more broadly representative cohort of donor parathyroid glands. 

The scientific objectives completed during the one-year project period are essential for comprehensive mapping of the human parathyroid gland.  The specific landmarks achieved include: demonstration that our live organ procurement work flow preserves tissue viability and maintains intact biochemical function; validation of recovery efficiency, parathyroid marker expression and cellular integrity in suspension; comparative assessment of whole cell vs nuclear isolation for downstream molecular analysis; validation of a novel split-pool sequencing approach that greatly improves capture efficiency, reduces selective recovery bias, and eliminates library construction batch effect concerns; digital spatial profiling of archived normal parathyroid gland sections to demonstrate the capture and whole transcriptome interrogation of specific cellular subsets demarcated by marker gene expression; and the molecular data from these studies showing that the cellular composition and transcriptional profiles of parathyroid gland tissue are dynamic rather than static.  This last finding reveals that the cellular content and biochemical activity of the parathyroid gland may be physiologically conditional, suggesting that functional reconstitution of the parathyroid gland is not a fixed target, but instead requires complementation of adaptive capacity in addition to terminally differentiated cellular phenotypes.  These key data will inform future and ongoing studies to reconstitute native parathyroid gland function.

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Publication:

Digital spatial profiling of human parathyroid tumors reveals cellular and molecular alterations linked to vitamin D deficiency

Chia-Ling Tu, Wenhan Chang, Julie A Sosa, James Koh

PNAS Nexus, Volume 2, Issue 3, March 2023, pgad073

Deciphering the structural consequences of ZC4H2 germline rare variants

Awardee: Prof. Michal Linial

Institution: The Hebrew University of Jerusalem

Award Amount: $50,000

Funding Period : January 1, 2021 - December 31, 2021

Studies on ZC4H2 to help better understand the pathophysiology of ZARD

Awardee: Vera Kalscheuer, PhD

Insitution: Max Planck Institute for Molecular Genetics

Award Amount: $50,000

Funding Period: January 1, 2021 - December 31, 2021

Nucleic acid binding by ZC4H2

Awardee: Daniel Dominguez, PhD

Institution: UNC at Chapel Hill

Award Amount: $50,000

Funding Period: January 1, 2021 - December 31, 2021

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Summary:

Mutations or genetic rearrangements in the protein, ZC4H2, cause a group of X-linked neurodevelopmental disorders for which there are no treatments. While the importance of this protein is clear, the specific function of ZC4H2 is still unknown. ZC4H2 is predicted to be a zinc-finger protein. We hypothesize that like many other zinc-finger proteins, ZC4H2 directly binds DNA or RNA, and functions to regulate gene expression programs required for normal development. Our goal is to determine if ZC4H2 interacts with nucleic acids and to identify specific genes and/or gene expression pathways that become dysfunctional when ZC4H2 is mutated. Patients suffering from ZC4H2-associated rare disorders have little recourse; understanding the biological function of this protein is a critical and necessary first step to uncover potential therapeutic approaches. 

A working prototype of an in-home ionized calcium monitoring device using a paper-based ion-selective optode and an optical reader

Awardee: Xuewei Wang, PhD

Institution: Virginia Commonwealth University

Award Amount: $149,019

Funding Period: January 1, 2020 - December 31, 2020

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Project Summary:

We are developing test strips for ionized calcium in finger-prick blood samples. One drop of blood can be easily introduced into the strip by patients. The optical response of the strip is recorded by a regular smartphone equipped with a customized app. The test can be finished within two minutes because of the fast sensor response. The concentration of ionized calcium can be accurately determined in a range of 0.1 to 5.0 mmol/L (0.4 to 20.0 mg/dL). There is no interference from other molecules and ions in the blood. Therefore, this new technology will enable the in-home measurement of calcium in the blood and allows the management of hypoparathyroidism by the patient themselves.

Final Summary:

Affordable and portable blood calcium sensors using a smartphone detector have been developed. These sensors empower patients to measure their calcium ion concentration at home using blood collected by fingerstick.

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Publications:

R. Wang, X. Wang. Sensing of inorganic ions in microfluidic devices. Sensors and Actuators B: Chemical 2021, 329, 129171

R. Wang, Y. Zhou, N. Ghanbari Ghalehjoughi, Y. Mawaldi, X. Wang. Ion-Induced Phase Transfer of Cationic Dyes for Fluorescence-Based Electrolyte Sensing in Droplet Microfluidics. Analytical Chemistry, 2021

N. Ghanbari Ghalehjoughi, R. Wang, S. Kelley, X. Wang. Ultrasensitive Ionophore-Based Liquid Sensors for Colorimetric Ion Measurements in Blood. Analytical Chemistry, 2023, 95, 12564-12564

Developing human pluripotent stem cells for investigation and treatment of hypoparathyroidism

Awardee: Rene Maehr, PhD

Institution: Umass Medical School

Award Amount: $500,000

Funding Period: January 1, 2020 -December 31, 2020

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Summary:

The parathyroid gland is critically involved in regulation of calcium homeostasis of the body. Hypoparathyroidism as encountered by parathyroid damage, hypoplasia, or as a result of thyroid and parathyroid surgery, results in chronic hypocalcemia and low-turnover bone disease. Human pluripotent stem cells could provide a virtually unlimited source of parathyroid-like cells with calcium level responsiveness, offering a unique opportunity for development of a cell replacement products capable of regulating calcium levels. To unlock human pluripotent stem cell-based treatment strategies, robust and safe stem cell differentiation protocols need to be established. Here, we propose to develop an approach that is based on human pluripotent stem cell differentiation according to a developmental roadmap, and cutting edge humanized mouse avatar models for functional evaluation of human parathyroid-like cells. We expect this rigorous approach to provide several high-impact resources, including a source of high-fidelity human parathyroid-like cells and novel mouse models for studying parathyroid function.

Publication:

Integration of single-cell transcriptomes and chromatin landscapes reveals regulatory programs driving pharyngeal organ development - Nature Communitcations

In vitro differentiation of parathyroid cells from stem cells

Awardee: Michael Mannstadt, MD

Institution: Massachusetts General Hospital/Harvard University

Award Amount: $1,000,000

Funding Period: January 1, 2020 - December 31, 2021

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Summary:

Parathyroid glands produce parathyroid hormone (PTH), which is necessary for regulating blood calcium and phosphate levels and maintaining bone health. Patients with insufficient parathyroid gland activity (hypoparathyroidism) can suffer from multiple symptoms caused by low blood calcium levels, including minor problems like muscle twitching or severe, life-threatening complications such as tetany and seizures. Conventional treatment with calcium and active vitamin D does not replace the functions of PTH and can lead to undesired long-term effects, such as kidney stones. PTH replacement therapy requires daily self-injections. 

Currently, testing of serum calcium involves a visit to a clinical laboratory, a blood draw, and a delay while the patient waits for a report of their test results. This delays dose adjustment and leads to hyper- or hypocalcemia.

The long-term goal of this proposal is to offer a regenerative therapy for patients with hypoparathyroidism using mature parathyroid cells differentiated from human stem cells.  With our collaborators from several institutions, including stem cell and developmental biologists, parathyroid surgeons, and specialists in microencapsulation of human stem cell-derived hormone-producing cells, we aim to define genetic mechanisms governing parathyroid cell fate specification during embryonic development.  We will target critical pathways using small molecule activators and inhibitors to facilitate parathyroid cell fate specification.  We will also test a novel microencapsulation technique for human parathyroid cells by transplantation in mice.