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Seq-ing Single Cells

NIDCR Postdoc Belinda R. Hauser sequences individual salivary gland cells and uses bioinformatics to explore the relationships between progenitor cell types

Belinda Hauser, PhDFor postdoctoral research fellow Belinda R. Hauser, PhD, a passion for science started with toy microscopes in elementary school. When other kids were playing with dolls and stuffed animals, she was peering into her big sister’s microscope to magnify the tiniest constituents of her home. That scientific toy opened up a world that no one else could see.

Today, both sisters are PhD biomedical scientists. Dr. Hauser fell in love with genetics, the study of the machinery that drives cellular processes, whereas her sister chose microbiology. A knack for science seems to be a heritable trait in her family: three female cousins completed graduate studies in biology as well.

Two years ago, senior investigator Matthew P. Hoffman, BDS, PhD, chief of NIDCR’s Matrix and Morphogenesis Section in NIH’s Intramural Research Program, invited Dr. Hauser to join his research lab. His team of scientists is identifying the cells, molecular signals, and other factors essential for salivary gland development and regeneration. They are targeting a number of cell types, including K5+ (keratin-5 positive) and K14+ (keratin-14 positive) progenitor cells, for potential use in salivary gland regeneration and repair.

At the time, Dr. Hauser was working in a dental research lab at Howard University in Washington, DC. For her dissertation project at Howard, she had used human cell lines and a tumor xenograft model to explore how miRNA-128 regulates tumor growth in head and neck cancer. When Dr. Hoffman visited Howard University to describe his work trying to regenerate salivary glands for head and neck cancer survivors, Dr. Hauser realized she could apply her microRNA sequencing skills to help identify gene regulatory networks useful for salivary gland regeneration. Naturally, applying her skills to this area of research appealed to her: one of the reasons she chose genetics for her life work was because she wanted to be able to identify the molecular mechanisms for diseases so that her discoveries could be used to help improve people’s health. If Dr. Hoffman’s team could regenerate glands, they could help cancer survivors who have dry mouth (xerostomia) caused by damage to salivary glands from radiation therapy. That work was something that she could feel passionate about.

“I always heard of NIH and how great the research is, but I never thought I would actually be here,” said Dr. Hauser. “It seemed like something so far away. …It was kind of intimidating, but I knew I could succeed here at NIH and grow as a scientist.”

Sifting through the haystack

Whereas other scientists in the lab are spying on cellular crosstalk to identify the molecular signals that cells rely on to communicate with each other during salivary gland development in an embryo, Dr. Hauser is running experiments to find out the relationship between K5+ and K14+ progenitor cells in the salivary gland in an adult.

“Our lab is interested in the regeneration of salivary glands after irradiation for head and neck cancer. We are interested in the K14;K5 subpopulation because our lab has shown the K14+ and K5+ cells are progenitors that give rise to ductal and epithelial compartments within the submandibular glands. We established that they are the progenitor cells but we don’t necessarily know the relation between them,” said Dr. Hauser. “My job is to characterize these individual cells of mice that express both K5 and K14. Is K14 dependent upon the K5, or is the K5 dependent upon the K14?”

To answer these questions, Dr. Hauser can’t rely on conventional assays. When the combined genetic material from a salivary gland containing various cell types is sequenced, it’s difficult to conclude which genes are expressed by which cell types. Instead, she divides the community of salivary gland cells into different populations of cells and uses high-throughput sequencing to obtain profiles and to analyze gene activity in individual cells. By isolating cell types and defining the functions, she can show how cells are related and may be able to predict how the gland will develop during regeneration.

“It’s kind of like finding that needle in the haystack,” said Dr. Hauser. “What’s unique about that, and how can we use that to regrow a gland to help a patient who is suffering from xerostomia.”

The difficult, two-phase process of running single cell analysis begins at 6:30 a.m., and she won’t head home until about 8:00 p.m. The first phase is isolation and enrichment. To prepare the sample, she needs to separate the salivary gland tissue into individual cells. She enriches for K5+, K14+, double positive (K5+;K14+), and double negative (K5-;K14-) cell populations using fluorescence-activated cell sorting (FACS). In the flow cytometer, the cells pass by in single file, a laser beam hits the cell, and the fluorescently tagged cell glows yellow for K5+;K14+, green for K5+, or red for K14+. Detectors pick up the fluorescence. Another detector picks up scattered lights from the cells. From these data, the flow cytometer obtains cell size and complexity for each cell that passes through.

For the second phase of capturing and preparing cells for sequencing and identification, Dr. Hauser uses microfluidics-based protocols, which rely on nanoscale chambers on a chip. She captures single cells on a microfluidic chip and creates libraries of RNA sequences to identify subtypes of cells.

“Salivary glands are so sensitive,” explained Dr. Hauser. “My goal is to capture 96 bright single cells.”

In September, Dr. Hauser received the 2017 NIH Fellows Award for Research Excellence for the abstract, “Single-cell RNA-seq analysis of adult salivary gland progenitors.” For this project, she sequenced 156 single-cell libraries with an average read depth of over one million reads. She was able to analyze the RNA expression of four subpopulations of cells: K5+, K14+, double positive (K5+;K14+), and double negative (K5-;K14-). She’s using bioinformatics, which can define groups based on gene variations, to determine the differences, if any, between these subpopulations.

Dr. Hauser has done multiple runs of the single cell analyses with the glands of mice that were about four months old, and she will soon study the cells of younger mice.

“The RNA seq will give you so much information that you have to sift through and see how it relates and which genes are expressed. That’s a lot of information. That’s the part I’m at now,” she said.

In September 2016, NIH Intramural Postdoctoral Fellows accepted the 2017 Fellows Award for Research Excellence. Only the top 25 percent of abstracts earn the FARE recognition, which comes with a $1,000 stipend to present the research results at a scientific meeting. Dr. Hauser appears in second row, third from the left in a white shirt.

From toy microscope to Zeiss optics

Dr. Hauser’s microscopy skills have advanced far beyond her youth mixing substances from around the home for magnification. She works with live cells—which have fluorescent markers and which are sensitive to changes in light and temperature—so her research relies on a special microscope that can detect fluorescence while controlling temperature and other variables.

To visualize each capture to ensure that she has obtained healthy cells that are fluorescently labeled, she uses an inverted Zeiss Axio Observer.Z1 research microscope equipped with a motorized stage. For access to this microscope and to the Fluidigm C1 system for helping prepare single cell libraries for sequencing​, she walks across Convent Drive from Building 30 to Building 35A to collaborate with Matthew W. Kelley, PhD, chief of the section on Developmental Neuroscience at the National Institute on Deafness and Other Communication Disorders (NIDCD), and his research fellow Michael C. Kelly, PhD.

In addition to sharing their research tools, Dr. Kelley and his postdoc Dr. Kelly have mentored Dr. Hauser and taught her how to run single cell sequencing. Because these NIDCD researchers are experts at this difficult work, they have also helped her troubleshoot during runs.

“My collaborators have been so helpful on this project. They are a godsend because there’s no way I could have done it alone,” she said. “With this microscope I can do a Z stack and check the fluorescence and see if there was a cell captured on the chip. Michael Kelly has helped me tremendously with the capture and imaging of single cells.”

A Z stack is a collection of images recorded at different focal planes —like a stack of extremely thin slices of onion. As Dr. Hauser slices down through the cell that is captured on the chip, she can focus on a fluorescent signal on an individual plane. At other planes, that signal may be out of focus. With a Z stack, she can analyze the entire cell.

Dr. Hauser said that she loves research and wants to continue to be a researcher after her postdoctoral fellowship is over. She likes being at NIH because of the collaborative spirit among scientists and the access to state-of-the-art research tools, scientific seminars, and classes. Most of all, she likes the freedom that Dr. Hoffman affords her to chart her own course. They meet every week to discuss progress and next steps, and he lets her figure out what she wants to do with her experiments.

“At a university, you’re kind of limited,” she said. “But here you can take your project a step further.”​​​

I knew I could succeed here at NIH and grow as a scienticst - Belinda Hauser, NIDCR Matris and Morphogenesis section. PhD, genetics and human genetics, Howard University, 2013.​​​​​​​​​​​​​​​​

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This page last updated: December 01, 2016