Human Skin Cells Converted into Embryonic Stem Cells by Cloning

The breakthrough marks the first time human stem cells have been produced via nuclear transfer and follows several unsuccessful attempts by research groups worldwide

Thursday, 16 May 2013

Scientists at Oregon Health & Science University and the Oregon National Primate Research Center (ONPRC) have successfully reprogrammed human skin cells to become embryonic stem cells capable of transforming into any other cell type in the body. It is believed that stem cell therapies hold the promise of replacing cells damaged through injury or illness. Diseases or conditions that might be treated through stem cell therapy include Parkinson's disease, multiple sclerosis, cardiac disease and spinal cord injuries.

The research breakthrough, led by Shoukhrat Mitalipov, Ph.D., a senior scientist at ONPRC, follows previous success in transforming monkey skin cells into embryonic stem cells in 2007. This latest research will be published in the journal Cell online May 15 and in print June 6.

Scematic description of how to create 

stem cells from human skin. Credit: 

ONPRC. 

The technique used by Drs. Mitalipov, Paula Amato, M.D., and their colleagues in OHSU's Division of Reproductive Endocrinology and Infertility, Department of Obstetrics & Gynecology, is a variation of a commonly used method called somatic cell nuclear transfer, or SCNT. It involves transplanting the nucleus of one cell, containing an individual's DNA, into an egg cell that has had its genetic material removed. The unfertilized egg cell then develops and eventually produces stem cells.

"Our finding offers new ways of generating stem cells for patients with dysfunctional or damaged tissues and organs," says senior study author Shoukhrat Mitalipov.

"Such stem cells can regenerate and replace those damaged cells and tissues and alleviate diseases that affect millions of people."

"A thorough examination of the stem cells derived through this technique demonstrated their ability to convert just like normal embryonic stem cells, into several different cell types, including nerve cells, liver cells and heart cells. Furthermore, because these reprogrammed cells can be generated with nuclear genetic material from a patient, there is no concern of transplant rejection," explained Dr. Mitalipov.

"While there is much work to be done in developing safe and effective stem cell treatments, we believe this is a significant step forward in developing the cells that could be used in regenerative medicine."

Another noteworthy aspect of this research is that it does not involve the use of fertilized embryos, a topic that has been the source of a significant ethical debate.

The first step during SCNT is enucleation

or removal of nuclear genetic material

(chromosomal) from a human egg. An

egg is positioned with holding pipette 

(on the left) and egg's chromosomes are

visualized under polarized microscope.

A hole is made in the egg's shell (zone

pellucida) using a laser and a smaller

pipette (on the right) is inserted through

the opening. The chromosomes then

sucked in inside the pipette and slowly

removed from the egg. Credit: Cell,

Tachibana et al.. 

The Mitalipov team's success in reprogramming human skin cells came through a series of studies in both human and monkey cells. In the past, researchers have used SCNT to generate only mouse and monkey embryonic stem cells — immature cells that can develop into different types of specialized cells, from neurons to heart muscle cells. Most previous attempts failed to produce human SCNT embryos that could progress beyond the 8-cell stage, falling far short of the 150-cell blastocyst stage that could provide hESCs for clinical purposes. Until now, it was not clear which factors and protocols are important for promoting SCNT embryonic development. Previous unsuccessful attempts by several labs showed that human egg cells appear to be more fragile than eggs from other species. Therefore, known reprogramming methods stalled before stem cells were produced.

To solve this problem, the OHSU group studied various alternative approaches first developed in monkey cells and then applied to human cells. Through moving findings between monkey cells and human cells, the researchers were able to develop a successful method.

The key to this success was finding a way to prompt egg cells to stay in a state called "metaphase" during the nuclear transfer process. Metaphase is a stage in the cell's natural division process (meiosis) when genetic material aligns in the middle of the cell before the cell divides. The research team found that chemically maintaining metaphase throughout the transfer process prevented the process from stalling and allowed the cells to develop and produce stem cells.

This is a colony of human ESCs (upper

portion) extracted from a blastocyst

generated by SCNT Credit: Cell,

Tachibana et al.. 

"This is a remarkable accomplishment by the Mitalipov lab that will fuel the development of stem cell therapies to combat several diseases and conditions for which there are currently no treatments or cures," said Dr. Dan Dorsa, Ph.D., OHSU Vice President for Research.

"The achievement also highlights OHSU's deep reproductive expertise across our campuses. A key component to this success was the translation of basic science findings at the OHSU primate centre paired with privately funded human cell studies."

One important distinction is that while the method might be considered a technique for cloning stem cells, commonly called therapeutic cloning, the same method would not likely be successful in producing human clones otherwise known as reproductive cloning. Several years of monkey studies that utilize somatic cell nuclear transfer have never successfully produced monkey clones. It is expected that this is also the case with humans. Furthermore, the comparative fragility of human cells as noted during this study is a significant factor that would likely prevent the development of clones.

"Our research is directed toward generating stem cells for use in future treatments to combat disease," added Dr. Mitalipov.

"While nuclear transfer breakthroughs often lead to a public discussion about the ethics of human cloning, this is not our focus, nor do we believe our findings might be used by others to advance the possibility of human reproductive cloning."

Source: Oregon Health & Science University 

Contact: Jim Newman and Mary Beth O'Leary (Cell Press)

Reference:

Human embryonic stem cells derived by somatic nuclear transfer

Masahito Tachibana, Paula Amato, Michelle Sparman, Nuria Marti Gutierrez, Rebecca Tippner-Hedges, Hong Ma, Eunju Kang, Alimujiang Fulati, Hyo-Sang Lee, Hathaitip Sritanaudomchai, Keith Masterson, Janine Larson, Deborah Eaton, Karen Sadler-Fredd, David Battaglia, David Lee, Diana Wu, Jeffrey Jensen, Phillip Patton, Sumita Gokhale, Richard L. Stouffer, Don Wolf, Shoukhrat Mitalipov

Cell, 15 May 2013, doi.org/10.1016/j.cell.2013.05.006, 2013

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Researchers Chart Epigenomic of Stem Cells that Mimic Early Human Development

Collaborative study will help overcome hurdles to using stem cells to treat diseases and injuries

Thursday, 09 May 2013

Scientists have long known that control mechanisms known collectively as "epigenetics" play a critical role in human development, but they did not know precisely how alterations in this extra layer of biochemical instructions in DNA contribute to development.

Now, in the first comprehensive analysis of epigenetic changes that occur during development, a multi-institutional group of scientists, including several from the Salk Institute for Biological Studies, has discovered how modifications in key epigenetic markers influence human embryonic stem cells as they differentiate into specialized cells in the body. The findings were published May 9 in Cell.

Professor Joseph R. Ecker, Plant Molecular and

Cellular Biology Laboratory, Howard Hughes

Medical Institute and Gordon and Betty Moore

Foundation Investigator, Salk International

Council Chair in Genetics. Credit:  Courtesy of 

the Salk Institute for Biological Studies.

"Our findings help us to understand processes that occur during early human development and the differentiation of a stem cell into specialized cells, which ultimately form tissues in the body," says co-lead author Joseph R. Ecker, a professor and director of Salk's Plant Molecular and Cellular Biology Laboratory and holder of the Salk International Council Chair in Genetics.

Scientists have established that the gene expression program encoded in DNA is carried out by proteins that bind to regulatory genes and modulate gene expression in response to environmental cues. Growing evidence now shows that maintenance of this process depends on epigenetic marks such as DNA methylation and chromatin modifications, biochemical processes that alter gene expression as cells divide and differentiate from embryonic stem cells into specific tissues. Epigenetic modifications - collectively known as the epigenome - control which genes are turned on or off without changing the letters of the DNA alphabet (A-T-C-G), providing cells with an additional tool to fine-tune how genes control the cellular machinery.

In their study, the Salk researchers and their collaborators from several prominent research institutions across the United States examined the beginning state of cells, before and after they developed into specific cell types. Starting with a single cell type-the H1 human embryonic stem cell - the most widely studied stem cell line to date - the team followed the cells' epigenome from development to different cell states, looking at the dynamics in changes to epigenetic marks from one state to another. Were they methylated, an essential process for normal development, or unmethylated?  

What happened to the cells during development? What regulatory processes occurred in the cell lineage?

The scientists found sections of the DNA that activate regulatory genes, which in turn control the activity of other genes, tend to have different amounts of letters of the DNA alphabet, "C" and "G" specifically, depending on when these regulatory genes are turned on during development.  

Additionally, regulatory genes that control early development are often located on stretches of DNA called methylation valleys, or DMVs, that are generally CG rich and devoid of epigenetic chemical modifications known as methylation. Consequently, these genes have to be regulated by another epigenetic mechanism, which the authors found were chemical changes called chromatin modifications. Chromatin is the mass of material-DNA and proteins-in a cell's nucleus that helps to control gene expression.

On the other hand, genes active in more mature cells whose tissue type is already determined tend to be CG poor and regulated by DNA methylation. The results suggest that distinct epigenetic mechanisms regulate early and late states of embryonic stem cell differentiation.

"Epigenomic studies of how stem cells differentiate into distinct cell types are a great way to understand early development of animals," says Ecker, who is also a Howard Hughes Medical Institute and Gordon and Betty Moore Foundation Investigator.

"If we understand how these cells' lineages originate, we can understand if something goes right or wrong during differentiation. It's a very basic study, but there are implications for being able to produce good quality cell types for various therapies."

For example, says Matthew Schultz, a graduate student in Ecker's lab, "understanding how development plays out normally could give us clues about how to reverse the process and turn normal adult cells into stem cells to regenerate tissues."

One area where the findings may help is in the study of tumour development. In normal tissue, DMVs are unmethylated, but in cancer, especially breast, colon and lung cancer, they are hypermethylated, suggesting, says Ecker, that alterations in the DNA methylation machinery might be an important mechanism aiding tumour development. He says further investigation is required to develop a greater understanding of this process.

Source: Salk Institute for Biological Studies

Contact: Andy Hoang

Reference:

Epigenomic Analysis of Multilineage Differentiation of Human Embryonic Stem Cells

Wei Xie, Matthew D. Schultz, Ryan Lister, Zhonggang Hou, Nisha Rajagopal, Pradipta Ray, John W. Whitaker, Shulan Tian, R. David Hawkins, Danny Leung, Hongbo Yang, Tao Wang, Ah Young Lee, Scott A. Swanson, Jiuchun Zhang, Yun Zhu, Audrey Kim, Joseph R. Nery, Mark A. Urich, Samantha Kuan, Chia-an Yen, Sarit Klugman, Pengzhi Yu, Kran Suknuntha, Nicholas E. Propson, Huaming Chen, Lee E. Edsall, Ulrich Wagner, Yan Li, Zhen Ye, Ashwinikumar Kulkarni, Zhenyu Xuan, Wen-Yu Chung, Neil C. Chi, Jessica E. Antosiewicz-Bourget, Igor Slukvin, Ron Stewart, Michael Q. Zhang, Wei Wang, James A. Thomson, Joseph R. Ecker, and Bing Ren
Cell, 09 May 2013, 10.1016/j.cell.2013.04.022

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Human Brain Cells Developed in Lab, Grow in Mice

Human Brain Cells Developed in Lab, Grow in Mice

Thursday, 09 May 2013

A key type of human brain cell developed in the laboratory grows seamlessly when transplanted into the brains of mice, UC San Francisco researchers have discovered, raising hope that these cells might one day be used to treat people with Parkinson's disease, epilepsy, and possibly even Alzheimer's disease, as well as and complications of spinal cord injury such as chronic pain and spasticity.

This is Arnold Kriegstein, M.D., Ph.D..

Credit: Susan Merrell/UCSF. 

"We think this one type of cell may be useful in treating several types of neurodevelopmental and neurodegenerative disorders in a targeted way," said Arnold Kriegstein, MD, PhD, director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF and co-lead author on the paper.

The researchers generated and transplanted a type of human nerve-cell progenitor called the medial ganglionic eminence (MGE) cell, in experiments described in the May 2 edition of Cell Stem Cell. Development of these human MGE cells within the mouse brain mimics what occurs in human development, they said.

Kriegstein sees MGE cells as a potential treatment to better control nerve circuits that become overactive in certain neurological disorders. Unlike other neural stem cells that can form many cell types — and that may potentially be less controllable as a consequence — most MGE cells are restricted to producing a type of cell called an interneuron. Interneurons integrate into the brain and provide controlled inhibition to balance the activity of nerve circuits.

To generate MGE cells in the lab, the researchers reliably directed the differentiation of human pluripotent stem cells — either human embryonic stem cells or induced pluripotent stem cells derived from human skin. These two kinds of stem cells have virtually unlimited potential to become any human cell type. When transplanted into a strain of mice that does not reject human tissue, the human MGE-like cells survived within the rodent forebrain, integrated into the brain by forming connections with rodent nerve cells, and matured into specialized subtypes of interneurons.

These findings may serve as a model to study human diseases in which mature interneurons malfunction, according to Kriegstein. The researchers' methods may also be used to generate vast numbers of human MGE cells in quantities sufficient to launch potential future clinical trials, he said.

Kriegstein was a co-leader of the research, along with Arturo Alvarez-Buylla, PhD, UCSF professor of neurological surgery; John Rubenstein, MD, PhD, UCSF professor of psychiatry; and UCSF postdoctoral scholars Cory Nicholas, PhD, and Jiadong Chen, PhD.

Nicholas utilized key growth factors and other molecules to direct the derivation and maturation of the human MGE-like interneurons. He timed the delivery of these factors to shape their developmental path and confirmed their progression along this path. Chen used electrical measurements to carefully study the physiological and firing properties of the interneurons, as well as the formation of synapses between neurons.

Previously, UCSF researchers led by Allan Basbaum, PhD, chair of anatomy at UCSF, have used mouse MGE cell transplantation into the mouse spinal cord to reduce neuropathic pain, a surprising application outside the brain. Kriegstein, Nicholas and colleagues now are exploring the use of human MGE cells in mouse models of neuropathic pain and spasticity, Parkinson's disease and epilepsy.

"The hope is that we can deliver these cells to various places within the nervous system that have been overactive and that they will functionally integrate and provide regulated inhibition," Nicholas said.

The researchers also plan to develop MGE cells from induced pluripotent stem cells derived from skin cells of individuals with autism, epilepsy, schizophrenia and Alzheimer's disease, in order to investigate how the development and function of interneurons might become abnormal — creating a lab-dish model of disease.

One mystery and challenge to both the clinical and pre-clinical study of human MGE cells is that they develop at a slower, human pace, reflecting an "intrinsic clock". In fast-developing mice, the human MGE-like cells still took seven to nine months to form interneuron subtypes that normally are present near birth.

"If we could accelerate the clock in human cells, then that would be very encouraging for various applications," Kriegstein said.

Source: University of California - San Francisco 

Contact: Jeffrey Norris

Reference:

Functional Maturation of hPSC-Derived Forebrain Interneurons Requires an Extended Timeline and Mimics Human Neural Development 

Cory R. Nicholas, Jiadong Chen, Yunshuo Tang, Derek G. Southwell, Nadine Chalmers, Daniel Vogt, Christine M. Arnold, Ying-Jiun J. Chen, Edouard G. Stanley, Andrew G. Elefanty, Yoshiki Sasai, Arturo Alvarez-Buylla, John L.R. Rubenstein, Arnold R. Kriegstein

Cell Stem Cell, Volume 12, Issue 5, 573-586, 2 May 2013, 0.1016/j.stem.2013.04.005

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