AI News, Robots grow mini-organs from human stem cells

Robots grow mini-organs from human stem cells

The advance promises to greatly expand the use of mini-organs in basic research and drug discovery, according to Benjamin Freedman, assistant professor of medicine, Division of Nephrology, at the UW School of Medicine, who led the research effort.

The traditional way to grow cells for biomedical research, Freeman explained, is to culture them as flat, two-dimensional sheets, which are overly simplistic.

In recent years, researchers have been increasingly successful in growing stem cells into more complex, three-dimensional structures called mini-organs or organoids.

Although similar approaches have been successful with adult stem cells, this is the first report of successfully automating the manufacture of organoids from pluripotent stem cells.

In this process, the liquid-handling robots introduced the stem cells into plates that contained as many as 384 miniature wells each, and then coaxed them to turn into kidney organoids over 21 days.

Harder and her colleagues at the University of Michigan Kidney Center used an automated, cutting-edge technique called single cell RNA sequencing to identify all the different types of cells found in the organoids.

In one of these experiments, they produced organoids with mutations that cause polycystic kidney disease, a common, inherited condition that affects one in 600 people worldwide and often leads to kidney failure.

They found that one, a factor called blebbistatin that blocks a protein called myosin, led to a significant increase in the number and size of cysts.

Robots grow mini-organs from human stem cells

An automated system that uses robots has been designed to rapidly produce human mini-organs derived from stem cells.

Researchers at the University of Washington School of Medicine in Seattle developed the new system.  The advance promises to greatly expand the use of mini-organs in basic research and drug discovery, according to Benjamin Freedman, assistant professor of medicine, Division of Nephrology, at the UW School of Medicine, who led the research effort.

In this process, the liquid-handling robots introduced the stem cells into plates that contained as many as 384 miniature wells each, and then coaxed them to turn into kidney organoids over 21 days.

With a speed that would have impressed Henry Ford's car assembly line, the robots could produce many plates in a fraction of the time.  'Ordinarily, just setting up an experiment of this magnitude would take a researcher all day, while the robot can do it in 20 minutes,' said Freedman.

'We established that these organoids do resemble developing kidneys, but also that they contain non-kidney cells that had not previously been characterized in these cultures,' said Harder.  'These findings give us a better idea of the nature of these organoids and provide a baseline from which we can make improvements,' Freedman said.

In one of these experiments, they produced organoids with mutations that cause polycystic kidney disease, a common, inherited condition that affects one in 600 people worldwide and often leads to kidney failure. In

The title of the research paper in Cell Stem Cell is, 'High-throughput screening enhances kidney organoid differentiation from human pluripotent stem cells and enables automated multidimensional phenotyping.'

High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping

Figure 7 Screening Reveals that Blebbistatin Increases PKD Organoid Cystogenesis (A) Cyst formation (% of cyst/organoid) from PKD organoids cultured in 96-well and treated with different compounds.

(C) Cyst quantification 3 days after blebbistatin treatment in suspension culture (n = 4 separate experiments, ≥15 organoids, ± SEM, p = 0.0002).

(D) Cyst diameters after 7 days of blebbistatin treatment in suspension culture from 4 separate experiments pooled together.

(E) Representative images and quantification of PKD cyst area after removal of blebbistatin (n = 8 from 2 separate experiments, ± SEM;

Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids

To evaluate the tissue-specific functions of undifferentiated hPSCs and descendant hPSC-KCs, we developed an adherent, 3D culture system for hPSCs that first produced epiblast spheroids and subsequently kidney tubules (Fig.

Spheroid cells exhibited polarized localization of podocalyxin (PODXL) to the luminal surface, zonula occludens 1 (ZO-1) to apical cell–cell junctions, and β-catenin (βCAT) to primarily basolateral membranes (Fig.

In nine serial passages, dissociated cavity-lining spheroid cells generated new cavitated spheroids after sandwiching, or alternatively flat colonies when the final passage was into monolayer conditions (Fig.

Even after extensive serial passages to/from 3D culture, cavity-lining cells implanted into immunodeficient mice efficiently produced teratoma tissues derived from all three embryonic germ layers (Fig.

Two-dimensional (2D) and 3D cultures exhibited similar growth rates, and pluripotency markers continued to be expressed in identical patterns in serially sandwiched cells, including octamer-binding transcription factor 4 (OCT4), sex-determining region Y box-2 (SOX2), NANOG and TRA-1-60 (Fig.

We hypothesized that hPSC spheroids model the epiblast epithelial mass, which forms a columnar epithelium surrounding an early amniotic cavity in human and primate implantation-stage embryos15,16.

Indeed, ‘naive’ hLR5 iPSCs, which form compact, ICM-like colonies similar to mouse (m)ESCs8, did not form lumens in 3D cultures even after 5 days of growth, whereas ‘primed’ hLR5-derived (LD-)iPSCs, which resemble epiblast-stage hPSCs, formed cavities efficiently in sandwich cultures (Supplementary Fig.

To differentiate epiblast spheroids into descendant epithelia, we applied a directed differentiation regimen originally designed for cardiomyocyte generation from 2D cultures, involving the sequential inhibition of glycogen synthase kinase-3β (GSK3β) and wingless-related integration site (WNT) signalling29.

Remarkably, rather than forming cardiomyocytes, spheroid cells underwent epithelial-to-mesenchymal transition to form a confluent monolayer that by day 10 aggregated into folds and initiated mesenchymal-to-epithelial transition into convoluted, translucent, tubular organoids (Fig.

Optimization of this new protocol revealed that spheroid formation, treatment with the GSK3β inhibitor Chiron Technologies 99021 (CHIR99021) and subsequent incubation in B27-supplemented media were sufficient to induce tubular differentiation, whereas insulin and WNT inhibition steps were dispensable (Supplementary Fig.

We furthermore observed anatomical progression of tubules from segments expressing E-cadherin, a distal tubule marker30, to LTL+ segments (proximal tubule), to capsule-like structures containing PODXL+ podocyte-like cells (Fig.

We therefore investigated the potential of kidney organoids to upregulate a clinical biomarker of proximal tubule injury, kidney injury molecule-1 (KIM-1), also known as T-cell immunoglobulin and mucin domain 1 and hepatitis A virus cellular receptor 1 (refs 34, 35).

To test whether epiblast spheroids and kidney tubules exhibit tissue-specific barrier functions, we developed a real-time assay to visualize molecular diffusion kinetics into and out of lumens, using fluorescent compounds of different sizes.

In epiblast spheroids, lucifer yellow (521 Da) added to the culture media for 2–4 h gradually accumulated within cavities, whereas rhodamine-conjugated dextran (RD, 10,000 Da) was excluded from lumens and instead accumulated in apical intercellular regions and formed a bright halo around the lumen (Fig.

When fluorescent compounds were incubated with spheroids for several hours and subsequently washed out, the compounds initially retained their distributions, but faded in intensity over time, indicating that they remained dynamic and were not fixed in location (Fig.

In contrast to epiblast spheroids, RD localized to tubular lumens in ∼80% of organoids after 2–4 h of incubation, and remained associated during a 24-h washout chase, without corresponding enrichment of lucifer yellow (Fig.

To investigate the functional role of podocalyxin in these cell types, podocalyxin knockout (PODXL−/−) hPSCs were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing system (Supplementary Fig.

Strikingly, compared with unmodified controls of otherwise identical genetic background, PODXL−/− hPSCs in 3D sandwich cultures exhibited a drastic (∼85%) decrease in their ability to produce hollow lumens, appearing instead as solid spheroids by phase contrast microscopy (Fig.

Although lumens were only very rarely observed by phase contrast microscopy in living cells, immunofluorescence in fixed cultures revealed small, compressed lumens in PODXL−/− hESC spheroids, lined with apically polarized ZO-1 (Supplementary Fig.

To investigate whether podocalyxin might contribute directly to luminal expansion via intermolecular charge repulsion, hPSCs were treated with a low concentration (8 μg ml−1) of protamine sulfate, a positively charged polycation that neutralizes the negatively charged, sialylated extracellular domain of podocalyxin41.

Both podocalyxin and another podocyte marker, Crumbs3, brightly coated the plasma membrane on the exterior of podocyte-like aggregates, whereas ZO-1, SYNPO and βCAT co-localized in a reciprocal pattern, forming internal zipper-like tracks between adjacent cell layers (Fig.

These structures remained tethered to the underlying matrix, but moved freely in response to vibration, in contrast to neighbouring tubular organoids that remained fixed in position near the surface of the dish (Supplementary Movie 2).

Culturing knowledge in stem cell research

Freedman is currently performing biomedical research using human pluripotent stem cells to model kidney disease pathophysiology and develop new therapies.

Scientists report that genome editing by CRISPR-Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway.

Researchers identified a new structure in human sperm that functions in the zygote and may provide new avenues for addressing male infertility and insights into early embryo developmental defects.

From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, scientists identified a prospectively separable CD33+CD34+CD38–CD45RA–CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles.

Mini-kidneys help scientists study disease

Benjamin Freedman of UW Medicine in Seattle talks about a study that used mini-kidney organoids to understand the progression of polycystic kidney disease.

Cell Shorts | Illuminating the kidney

In our new series "Cell Shorts," see how Beno Freedman and his colleagues at the University of Washington are using our publicly available human induced ...

Support NKF Research

Support NKF Research: Benjamin Freedman, PhD, discusses his breakthroughs creating kidney organoids, with the ..

Discovery Gala 2016 - Northwest Kidney Centers

Russ and Yvette Stepp share their transplant story and discuss the impact polycystic kidney disease has on families. Dr. Beno Freedman explains how his team ...

Machines now able to produce MINI-HUMAN organs in huge breakthrough for health

Experts from the University of Washington School of Medicine have created a machine which can produce mini-organs of humans from stem cells.The ability to ...

Developing an Artificial Kidney - Part 3 with Dr. Morteza Ahmadi

Implantable artificial kidney may cure chronic kidney disease

About 85000 people need kidney transplants each year, but fewer than 20000 kidneys are available. To survive, people with chronic kidney disease must ...

Lab Grown Brains!

A recent scientific breakthrough has given scientists the ability to grow a tiny human brain in a lab. The mini brains are called organoids and though they are not ...

Robots to be used grow HUMAN ORGANS in freaky experiments

The automated machines have been designed to grow the organs using human stem cells in just 21 days.However, the organs are not actually full-sized and are ...

Stressed out: a novel approach to cancer immunotherapy

Stressed out: a novel approach to cancer immunotherapy Air date: Monday, November 7, 2016, 3:00:00 PM Category: WALS - Wednesday Afternoon Lectures ...