Posts Tagged ‘Stem Cell Biology and Regenerative Medicine’

A new type of Stem Cell

Larry H. Bernstein, MD, FCAP, LPBI



New Type of Stem Cell Discovered


Researchers at Michigan State University say they have discovered a new kind of stem cell, one that could lead to advances in regenerative medicine as well as offer new ways to study birth defects and other reproductive problems. Tony Parenti, lead author and MSU cell and molecular biology graduate student, unearthed the new cells, induced XEN cells, or iXEN, in a cellular trash pile, of sorts. The research is described in Stem Cell Reports.

“Other scientists may have seen these cells before, but they were considered to be defective, or cancer-like,” said Parenti, who works in the lab of Amy Ralston, Ph.D., MSU biochemist, cell and molecular biologist and co-author of the study. “Rather than ignore these cells that have been mislabeled as waste byproducts, we found gold in the garbage.”

A great deal of stem cell research focuses on new ways to make and use pluripotent stem cells. Pluripotent stem cells can be created by reactivating embryonic genes to “reprogram” mature adult cells. Reprogramming mature cells into induced pluripotent stem cells, or iPSCs, allows them to become malleable building blocks that can morph into any cell in the body.

For example, if a patient has a defective liver, healthy cells could be taken from the patient and reprogrammed into iPSCs, which could then be used to help regenerate the person’s failing organ. Taking cells from the same patient may greatly reduce the chance of the body rejecting the new treatment, Parenti said.

Prior to the discovery of reprogramming, scientists developed pluripotent stem cells from embryos. However, the embryo produces not only pluripotent stem cells, but also XEN cells, a stem cell type with unique properties. While pluripotent stem cells produce cells in the body, XEN cells produce extraembryonic tissues that play an essential but indirect role in fetal development.

Parenti and his team speculated that if the embryo produces both pluripotent and XEN cells, this might also occur during reprogramming. The eureka moment came when Parenti discovered colonies of iXEN cells popping up like weeds in his iPSC cultures. Using mice models, the team spent 6 months proving that these genetic weeds are not cancer-like, as previously suspected, but in fact, a new kind of stem cell with desirable properties.

Even more surprising, the team found that by inhibiting expression of XEN genes during reprogramming, they could decrease production of iXEN cells and increase production of iPSCs.

“Nature makes stem cells perfectly, but we are still trying to improve our stem cell production,” Parenti said. “We took what we learned by studying the embryo and applied it to reprogramming, and this opened up a new way to optimize reprogramming.”

The next steps of this research will involve seeing if this process occurs in human cells. XEN cells have yet to be discovered in humans, but the possibility of their existence is a key focus of the field.

“It’s a missing tool that we don’t have yet,” Dr. Ralston said. “It’s true that XEN cells have characteristics that pluripotent stem cells do not have. Because of those traits, iXEN cells can shed light on reproductive diseases. If we can continue to unlock the secrets of iXEN cells, we may be able to improve induced pluripotent stem cell quality and lay the groundwork for future research on tissues that protect and nourish the human embryo.”

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Adipocyte Derived Stroma Cells: Their Usage in Regenerative Medicine and Reprogramming into Pancreatic Beta-Like Cells

Curator: Evelina Cohn, Ph.D.

The following presentation can be dowloaded in PowerPoint form by clicking on the link below:

adipocytes (1)


In Summary:

There are different results related to betatrophin and its characteristic to induce insulin and/or expand the pancreas beta cells. All the experiments so far were performed in mice. Some of the authors like Elisabeth Kugelberg from Harvard University agrees that betatrophin can induce insulin and expansion of secreting beta cells in mice (E. Kugelberg , 2014). Levitsky et al., 2014, come to the conclusion that betatrophin stimulate growth of beta cells in mice, while Gusarova et al., 2014, said that Betatrophin doesn’t control cell expansion in mice ( Gusarova et al., 2014) All three results are based on experiments on mice.

To make sure what are the characteristics of betatrophin in human pancreatic beta cells I suggest to try to determine the concentration and effect on those concentrations on immortal beta cells from human, CM cell line (insulinoma-obtained from ascitic fluid of cancer patients ) ( they are not producing any insulin under the glucose stimulation, therefore they may be a good for our model if they respond to betatrophin) TRM-1 (foetal Human SV40 T antigen)-Express small amount of insulin, not responsive to glucose stimulation) and finally Blox5 ( foetal Human SV40 T –antigen) which Exhibit glucose responsive. and Low insulin content. Blox5 may be the second good cell line to experiment, because they are responsive to glucose and they may be responsive to betatrophin as well.

If we found that those cell lines are inducing insulin then we may try primary beta cells. There is an article of 2013 (Ilie and Ilie, 2013) in which there is a possibility of regeneration of beta cells in vivo by neogenesis from adult pancreas. We can use their model to see if betatrophin indeed induce insulin in those cells. ( see the article attached)

On the other hand there are possibilities of growing beta cells directly onto pancreatic duct as it shows below:

pharmacoogicalapproaches to islet regeneration











From: https://infodiabet.wordpress.com/2010/08/31/new-sources-of-pancreatic-beta-cells/

Therefore, I suggest of producing pancreatic duct by using 3D printing and grow the cells by neogenesis

directly on the pancreatic duct.


Gusarova V, Alexa CA, Na E, Stevis PE, Xin Y, Bonner-Weir S,

Cohen JC, Hobbs HH, Murphy AJ, Yancopoulos GD, Gromada J (2014), ANGPTL8/Betatrophin Does Not Control Pancreatic Beta Cell Expansion. Cell 159: 691-696.

Kugelberg E. (2013) Diabetes: Betatrophin—inducing β-cell expansion to treat diabetes mellitus? Nature Reviews Endocrinology 9: 379

Levitsky LL, Ardestani G, Rhoads DB (2014). Role of growth factors in control of pancreatic beta cell mass: focus on betatrophin. Curr Opin Pediatr. August 26 (4):475-9










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Previously unseen immune reaction identified for stem cell transplants.

Reporter: Stephen J. Williams, Ph.D.

Reposted from at http://health-innovations.org/2014/11/21/previously-unseen-immune-reaction-identified-in-stem-cell-transplants/


Mouse cells and tissues created through nuclear transfer can be rejected by the body because of a previously unknown immune response to the cell’s mitochondria, according to an international study in mice by researchers at the Stanford University, MIT and colleagues in Germany and England.  The findings reveal a likely, but surmountable, hurdle if such therapies are ever used in humans, the researchers said.  The opensource study is published in Cell Stem Cell.

Stem cell therapies hold vast potential for repairing organs and treating disease. The greatest hope rests on the potential of pluripotent stem cells, which can become nearly any kind of cell in the body. One method of creating pluripotent stem cells is called somatic cell nuclear transfer, and involves taking the nucleus of an adult cell and injecting it into an egg cell from which the nucleus has been removed.

The promise of the SCNT method is that the nucleus of a patient’s skin cell, for example, could be used to create pluripotent cells that might be able to repair a part of that patient’s body.  One attraction of SCNT has always been that the genetic identity of the new pluripotent cell would be the same as the patient’s, since the transplanted nucleus carries the patient’s DNA.

The hope has been that this would eliminate the problem of the patient’s immune system attacking the pluripotent cells as foreign tissue, which is a problem with most organs and tissues when they are transplanted from one patient to another.

Stanford University have raised the possibility in the past that the immune system of a patient who received SCNT-derived cells might still react against the cells’ mitochondria, which act as the energy factories for the cell and have their own DNA. This reaction could occur because cells created through SCNT contain mitochondria from the egg donor and not from the patient, and therefore could still look like foreign tissue to the recipient’s immune system.

That hypothesis was never tested until the team took up the challenge.  There was a thought that because the mitochondria were on the inside of the cell, they would not be exposed to the host’s immune system.  The current study found that this was not the case.

The team used cells that were created by transferring the nuclei of adult mouse cells into enucleated eggs cells from genetically different mice. When transplanted back into the nucleus donor strain, the cells were rejected although there were only two single nucleotide substitutions in the mitochondrial DNA of these SCNT-derived cells compared to that of the nucleus donor.  The team were surprised to find that just two small differences in the mitochondrial DNA was enough to cause an immune reaction.

Until recently, researchers were able to perform SCNT in many species, but not in humans. When scientists at the Oregon Health and Science University announced success in performing SCNT with human cells last year, it reignited interest in eventually using the technique for human therapies. Although many stem cell researchers are focused on a different method of creating pluripotent stem cells, called induced pluripotent stem cells, there may be some applications for which SCNT-derived pluripotent cells are better suited.

The immunological reactions reported in the new paper will be a consideration if clinicians ever use SCNT-derived stem cells in human therapy, but such reactions should not prevent their use.   This research informs the medical community of the margin of safety that would be required if, in the distant future, researchers need to use SCNT to create pluripotent cells to treat someone.  In that case, clinicians would likely be able to handle the immunological reaction using the immunosuppression methods that are currently available.

In the future, scientists might also lessen the immune reaction by using eggs from someone who is genetically similar to the recipient, such as a mother or sister.

Source:  Stanford University School of Medicine

The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies.  SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts.  Schrepfer et al 2014.

The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies. SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts. Schrepfer et al 2014.

SCNT (somatic cell nuclear transfer)


Possible ways to generate immune-compatible derivatives of pluripotent cells. From Nature Reviews

From the following article: Derive and conquer: sourcing and differentiating stem cells for therapeutic applications

In genetics and developmental biology, somatic cell nuclear transfer (SCNT) is a laboratory technique for creating an ovum with a donor nucleus. It can be used in embryonic stem cell research, or in regenerative medicine where it is sometimes referred to as “therapeutic cloning.”

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New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue

Reporter: Stephen J. Williams, Ph.D.

The FDA recently came out with a Draft Guidance on use of human cells, tissues and cellular and tissue-based products (HCT/P) {defined in 21 CFR 1271.3(d)} and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.

A full copy of the PDF can be found here for reference but the following is a summary of points of the guidance.FO508ver – 2015-373 HomologousUseGuidanceFinal102715

In 21 CFR 1271.10, the regulations identify the criteria for regulation solely under section 361 of the PHS Act and 21 CFR Part 1271. An HCT/P is regulated solely under section 361 of the PHS Act and 21 CFR Part 1271 if it meets all of the following criteria (21 CFR 1271.10(a)):

  • The HCT/P is minimally manipulated;
  • The HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent;
  • The manufacture of the HCT/P does not involve the combination of the cells or tissues with another article, except for water, crystalloids, or a sterilizing, preserving, or storage agent, provided that the addition of water, crystalloids, or the sterilizing, preserving, or storage agent does not raise new clinical safety concerns with respect to the HCT/P; and
  • Either:
  1. The HCT/P does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function; or
  2. The HCT/P has a systemic effect or is dependent upon the metabolic activity of living cells for its primary function, and:
  3. Is for autologous use;
  4. Is for allogeneic use in a first-degree or second-degree blood relative; or
  5. Is for reproductive use.

If an HCT/P does not meet all of the criteria in 21 CFR 1271.10(a), and the establishment that manufactures the HCT/P does not qualify for any of the exceptions in 21 CFR 1271.15, the HCT/P will be regulated as a drug, device, and/or biological product under the Federal Food, Drug and Cosmetic Act (FD&C Act), and/or section 351 of the PHS Act, and applicable regulations, including 21 CFR Part 1271, and pre-market review will be required.

1 Examples of HCT/Ps include, but are not limited to, bone, ligament, skin, dura mater, heart valve, cornea, hematopoietic stem/progenitor cells derived from peripheral and cord blood, manipulated autologous chondrocytes, epithelial cells on a synthetic matrix, and semen or other reproductive tissue. The following articles are not considered HCT/Ps: (1) Vascularized human organs for transplantation; (2) Whole blood or blood components or blood derivative products subject to listing under 21 CFR Parts 607 and 207, respectively; (3) Secreted or extracted human products, such as milk, collagen, and cell factors, except that semen is considered an HCT/P; (4) Minimally manipulated bone marrow for homologous use and not combined with another article (except for water, crystalloids, or a sterilizing, preserving, or storage agent, if the addition of the agent does not raise new clinical safety concerns with respect to the bone marrow); (5) Ancillary products used in the manufacture of HCT/P; (6) Cells, tissues, and organs derived from animals other than humans; (7) In vitro diagnostic products as defined in 21 CFR 809.3(a); and (8) Blood vessels recovered with an organ, as defined in 42 CFR 121.2 that are intended for use in organ transplantation and labeled “For use in organ transplantation only.” (21 CFR 1271.3(d))

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Draft – Not for Implementation

Section 1271.10(a)(2) (21 CFR 1271.10(a)(2)) provides that one of the criteria for an HCT/P to be regulated solely under section 361 of the PHS Act is that the “HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.” As defined in 21 CFR 1271.3(c), homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor. This criterion reflects the Agency’s conclusion that there would be increased safety and effectiveness concerns for HCT/Ps that are intended for a non-homologous use, because there is less basis on which to predict the product’s behavior, whereas HCT/Ps for homologous use can reasonably be expected to function appropriately (assuming all of the other criteria are also met).2 In applying the homologous use criterion, FDA will determine what the intended use of the HCT/P is, as reflected by the the labeling, advertising, and other indications of a manufacturer’s objective intent, and will then apply the homologous use definition.

FDA has received many inquiries from manufacturers about whether their HCT/Ps meet the homologous use criterion in 21 CFR 1271.10(a)(2). Additionally, transplant and healthcare providers often need to know this information about the HCT/Ps that they are considering for use in their patients. This guidance provides examples of different types of HCT/Ps and how the regulation in 21 CFR 1271.10(a)(2) applies to them, and provides general principles that can be applied to HCT/Ps that may be developed in the future. In some of the examples, the HCT/Ps may fail to meet more than one of the four criteria in 21 CFR 1271.10(a).


  1. What is the definition of homologous use?

Homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor (21 CFR 1271.3(c)), including when such cells or tissues are for autologous use. We generally consider an HCT/P to be for homologous use when it is used to repair, reconstruct, replace, or supplement:

  • Recipient cells or tissues that are identical (e.g., skin for skin) to the donor cells or tissues, and perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor; or,
  • Recipient cells that may not be identical to the donor’s cells, or recipient tissues that may not be identical to the donor’s tissues, but that perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor.3

2 Proposed Approach to Regulation of Cellular and Tissue-Based Products, FDA Docket. No. 97N-0068 (February. 28, 1997) page 19. http://www.fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/tissue/ ucm062601.pdf.

3“Establishment Registration and Listing for Manufacturers of Human Cellular and Tissue-Based Products” 63 FR 26744 at 26749 (May 14, 1998).

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1-1. A heart valve is transplanted to replace a dysfunctional heart valve. This is homologous use because the donor heart valve performs the same basic function in the donor as in the recipient of ensuring unidirectional blood flow within the heart.

1-2. Pericardium is intended to be used as a wound covering for dura mater defects. This is homologous use because the pericardium is intended to repair or reconstruct the dura mater and serve as a covering in the recipient, which is one of the basic functions it performs in the donor.

Generally, if an HCT/P is intended for use as an unproven treatment for a myriad of

diseases or conditions, the HCT/P is likely not intended for homologous use only.4

  1. What does FDA mean by repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues?

Repair generally means the physical or mechanical restoration of tissues, including by covering or protecting. For example, FDA generally would consider skin removed from a donor and then transplanted to a recipient in order to cover a burn wound to be a homologous use. Reconstruction generally means surgical reassembling or re-forming. For example, reconstruction generally would include the reestablishment of the physical integrity of a damaged aorta.5 Replacement generally means substitution of a missing tissue or cell, for example, the replacement of a damaged or diseased cornea with a healthy cornea or the replacement of donor hematopoietic stem/progenitor cells in a recipient with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. Supplementation generally means to add to, or complete. For example, FDA generally would consider homologous uses to be the implantation of dermal matrix into the facial wrinkles to supplement a recipient’s tissues and the use of bone chips to supplement bony defects. Repair, reconstruction, replacement, and supplementation are not mutually exclusive functions and an HCT/P could perform more than one of these functions for a given intended use.

  1. What does FDA mean by “the same basic function or functions” in the definition of homologous use?

For the purpose of applying the regulatory framework, the same basic function or functions of HCT/Ps are considered to be those basic functions the HCT/P performs in the body of the donor, which, when transplanted, implanted, infused, or transferred, the HCT/P would be expected to perform in the recipient. It is not necessary for the HCT/P in the recipient to perform all of the basic functions it performed in the donor, in order to

4 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).

5 “Current Good Tissue Practice for Human Cell, Tissue, and Cellular and Tissue-Based Product Establishments; Inspection and Enforcement” 69 FR 68612 at 68643 (November 24, 2004) states, “HCT/Ps with claims for “reconstruction or repair” can be regulated solely under section 361 of the PHS Act, provided the HCT/P meets all the criteria in § 1271.10, including minimal manipulation and homologous use.”

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meet the definition of homologous use. However, to meet the definition of homologous use, any of the basic functions that the HCT/P is expected to perform in the recipient must be a basic function that the HCT/P performed in the donor.

A homologous use for a structural tissue would generally be to perform a structural function in the recipient, for example, to physically support or serve as a barrier or conduit, or connect, cover, or cushion.

A homologous use for a cellular or nonstructural tissue would generally be a metabolic or biochemical function in the recipient, such as, hematopoietic, immune, and endocrine functions.

3-1. The basic functions of hematopoietic stem/progenitor cells (HPCs) include to form and to replenish the hematopoietic system. Sources of HPCs include cord blood, peripheral blood, and bone marrow.6

  1. HPCs derived from peripheral blood are intended for transplantation into an individual with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. This is homologous use because the peripheral blood product performs the same basic function of reconstituting the hematopoietic system in the recipient.
  2. HPCs derived from bone marrow are infused into an artery with a balloon catheter for the purpose of limiting ventricular remodeling following acute myocardial infarction. This is not homologous use because limiting ventricular remodeling is not a basic function of bone marrow.
  3. A manufacturer provides HPCs derived from cord blood with a package insert stating that cord blood may be infused intravenously to differentiate into neuronal cells for treatment of cerebral palsy. This is not homologous use because there is insufficient evidence to support that such differentiation is a basic function of these cells in the donor.

3-2. The basic functions of the cornea include protecting the eye by forming its outermost layer and serving as the refracting medium of the eye. A corneal graft is transplanted to restore sight in a patient with corneal blindness. This is homologous use because a corneal graft performs the same basic functions in the donor as in the recipient.

3-3. The basic functions of a vein or artery include serving as a conduit for blood flow throughout the body. A cryopreserved vein or artery is used for arteriovenous access during hemodialysis. This is homologous use because the vein or artery is supplementing the vessel as a conduit for blood flow.

3-4. The basic functions of amniotic membrane include covering, protecting, serving as a selective barrier for the movement of nutrients between the external and in utero

6 Bone marrow meets the definition of an HCT/P only if is it more than minimally manipulated; intended by the manufacturer for a non-homologous use, or combined with certain drugs or devices.

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environment, and to retain fluid in utero. Amniotic membrane is used for bone tissue replacement to support bone regeneration following surgery to repair or replace bone defects. This is not a homologous use because bone regeneration is not a basic function of amniotic membrane.

3-5. The basic functions of pericardium include covering, protecting against infection, fixing the heart to the mediastinum, and providing lubrication to allow normal heart movement within chest. Autologous pericardium is used to replace a dysfunctional heart valve in the same patient. This is not homologous use because facilitating unidirectional blood flow is not a basic function of pericardium.

  1. Does my HCT/P have to be used in the same anatomic location to perform the same basic function or functions?

An HCT/P may perform the same basic function or functions even when it is not used in the same anatomic location where it existed in the donor.7 A transplanted HCT/P could replace missing tissue, or repair, reconstruct, or supplement tissue that is missing or damaged, either when placed in the same or different anatomic location, as long as it performs the same basic function(s) in the recipient as in the donor.

4-1. The basic functions of skin include covering, protecting the body from external force, and serving as a water-resistant barrier to pathogens or other damaging agents in the external environment. The dermis is the elastic connective tissue layer of the skin that provides a supportive layer of the integument and protects the body from mechanical stress.

  1. An acellular dermal product is used for supplemental support, protection, reinforcement, or covering for a tendon. This is homologous use because in both anatomic locations, the dermis provides support and protects the soft tissue structure from mechanical stress.
  2. An acellular dermal product is used for tendon replacement or repair. This is not homologous use because serving as a connection between muscle and bone is not a basic function of dermis.

4-2. The basic functions of amniotic membrane include serving as a selective barrier for the movement of nutrients between the external and in utero environment and to retain fluid in utero. An amniotic membrane product is used for wound healing of dermal ulcers and defects. This is not homologous use because wound healing of dermal lesions is not a basic function of amniotic membrane.

4-3. The basic functions of pancreatic islets include regulating glucose homeostasis within the body. Pancreatic islets are transplanted into the liver through the portal vein,

7 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).


Contains Nonbinding Recommendations
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for preservation of endocrine function after pancreatectomy. This is homologous use because the regulation of glucose homeostasis is a basic function of pancreatic islets.

  1. What does FDA mean by “intended for homologous use” in 21 CFR 1271.10(a)(2)?

The regulatory criterion in 21 CFR 1271.10(a)(2) states that the HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.

Labeling includes the HCT/P label and any written, printed, or graphic materials that supplement, explain, or are textually related to the product, and which are disseminated by or on behalf of its manufacturer.8 Advertising includes information, other than labeling, that originates from the same source as the product and that is intended to supplement, explain, or be textually related to the product (e.g., print advertising, broadcast advertising, electronic advertising (including the Internet), statements of company representatives).9

An HCT/P is intended for homologous use when its labeling, advertising, or other indications of the manufacturer’s objective intent refer to only homologous uses for the HCT/P. When an HCT/P’s labeling, advertising, or other indications of the manufacturer’s objective intent refer to non-homologous uses, the HCT/P would not meet the homologous use criterion in 21 CFR 1271.10(a)(2).

  1. What does FDA mean by “manufacturer’s objective intent” in 21 CFR 1271.10(a)(2)?

A manufacturer’s objective intent is determined by the expressions of the manufacturer or its representatives, or may be shown by the circumstances surrounding the distribution of the article. A manufacturer’s objective intent may, for example, be shown by labeling claims, advertising matter, or oral or written statements by the manufacturer or its representatives. It may be shown by the circumstances that the HCT/P is, with the knowledge of the manufacturer or its representatives, offered for a purpose for which it is neither labeled nor advertised.

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Noncanonical Neural Stem Cell Signaling Pathways

Larry H. Bernstein, MD, FCAP, Curator



Concise Review: Reprogramming, Behind the Scenes: Noncanonical Neural Stem Cell Signaling Pathways Reveal New, Unseen Regulators of Tissue Plasticity With Therapeutic Implications


Interest is great in the new molecular concepts that explain, at the level of signal transduction, the process of reprogramming. Usually, transcription factors with developmental importance are used, but these approaches give limited information on the signaling networks involved, which could reveal new therapeutic opportunities. Recent findings involving reprogramming by genetic means and soluble factors with well-studied downstream signaling mechanisms, including signal transducer and activator of transcription 3 (STAT3) and hairy and enhancer of split 3 (Hes3), shed new light into the molecular mechanisms that might be involved. We examine the appropriateness of common culture systems and their ability to reveal unusual (noncanonical) signal transduction pathways that actually operate in vivo. We then discuss such novel pathways and their importance in various plastic cell types, culminating in their emerging roles in reprogramming mechanisms. We also discuss a number of reprogramming paradigms (mouse induced pluripotent stem cells, direct conversion to neural stem cells, and in vivo conversion of acinar cells to β-like cells). Specifically for acinar-to-β-cell reprogramming paradigms, we discuss the common view of the underlying mechanism (involving the Janus kinase-STAT pathway that leads to STAT3-tyrosine phosphorylation) and present alternative interpretations that implicate STAT3-serine phosphorylation alone or serine and tyrosine phosphorylation occurring in sequential order. The implications for drug design and therapy are important given that different phosphorylation sites on STAT3 intercept different signaling pathways. We introduce a new molecular perspective in the field of reprogramming with broad implications in basic, biotechnological, and translational research.


Reprogramming is a powerful approach to change cell identity, with implications in both basic and applied biology. Most efforts involve the forced expression of key transcription factors, but recently, success has been reported with manipulating signal transduction pathways that might intercept them. It is important to start connecting the function of the classic reprogramming genes to signaling pathways that also mediate reprogramming, unifying the sciences of signal transduction, stem cell biology, and epigenetics. Neural stem cell studies have revealed the operation of noncanonical signaling pathways that are now appreciated to also operate during reprogramming, offering new mechanistic explanations.


Progress in biomedical science has been hindered by the all too common difficulty in translating in vitro observations to in vivo systems [1]. This problem applies to both transformed and primary cell culture systems, suggesting a difficulty in modeling the in vivo signaling state of a cell inside a culture dish. Traditionally, highly cancerous or genetically altered cells have been used in research, because they are typically easier to grow than primary cells. Serum is often included in the culture medium as a generic growth stimulator, providing a plethora of undefined nutrients and signal transduction pathway modulators. However, the propensity of primary cells to irreversibly differentiate in these conditions has forced experimentalists to modify how these cells are maintained in vitro, removing serum and using specific mitogens, to preserve their self-renewal state [2]. The value of such culture systems is not merely a practical one allowing for their expansion, they also force cells to grow using particular signaling pathways (that promote self-renewal) at the expense of others (that promote differentiation) and can serve as formidable model systems by providing access to these pathways. Early indications revealed that a variety of immature and differentiated plastic cells use common, noncanonical signaling pathways, with implications in regenerative medicine, cancer, diabetes, and reprogramming technologies.

An example of a signaling pathway that can be identified through this rationale is the STAT3-Ser/Hes3 signaling axis. It was originally characterized in neural stem cells (NSCs) and subsequently shown to regulate additional cell types, including prostate tumor-initiating cells, glioblastoma multiforme cancer stem cells (GBM CSCs), adrenomedullary chromaffin progenitors, and mouse insulinoma cell lines (Fig. 1A) [37]. In brief, at the center of the pathway is the phosphorylation of STAT3 on serine residue 727 (STAT3-Ser) [8]. This modification is largely redundant for many cell types but is of great importance to the survival of NSCs. STAT3-Ser is a convergence point for several other stimuli, including fibroblast growth factor (FGF), a noncanonical Notch signaling branch, the angiopoietin2/Tie2 system, and insulin. These lead to STAT3-Ser phosphorylation and subsequent transcriptional activation of Hes3, a transcription factor and passive repressor, with roles that are only now starting to be understood [911]. The pathway is opposed by Janus kinase (JAK) activity, a key component of the growth machinery of many cell types [12]. In addition to NSCs, pharmacological inhibitors of JAK promote the survival of human pluripotent stem cells and the developmentally equivalent mouse equivalent epiblast stem cell, further highlighting the stark differences in signal transduction preferences between most cell types studied and stem cell populations [13, 14].

Figure 1.

Noncanonical signaling pathway regulation during reprogramming. (A):Extracellular factors lead to the phosphorylation of STAT3-Tyr via JAK activation or STAT3-Ser via MAPK, Akt, and mTOR activation, and subsequent Hes3 transcription. The two pathways are opposing (e.g., JAK activity in neural stem cells [NSCs] suppresses induction of Hes3). Some cell types (e.g., primary NSCs) are confined to using the STAT3-Ser branch, because the STAT3-Tyr branch leads to their irreversible differentiation. Other cell types (e.g., primary cancer stem cells from glioblastoma multiforme patients and MIN6 cells) grow effectively using either pathway and, through repeated changes in cell culture conditions, can switch their signaling state back and forth. (B): Genes in the STAT3-Ser/Hes3 signaling axis are regulated during mouse fibroblast reprogramming. Sox21, Hes3, and Shh gene expression increases as MEFs transition to SSEA1+ and then to Oct4+ populations during reprogramming to the pluripotent state. Hes3 and Shh are downregulated in resultant stable mouse iPS cells grown in culture conditions that activate JAK (lines not to scale; expression levels at the MEF stage normalized to help visualize patterns and trends). (C): Genes in the STAT3-Ser/Hes3 signaling axis are regulated during neural specification of hES cells. The diagram summarizes the expression patterns of Hes3, Bmi1, and JAK1 over the course of a 77-day protocol to differentiate the human ES cell line WA09 to dorsal telencephalic neuronal fates (lines not to scale; expression levels at day 0 of ES cell stage normalized to help visualize patterns and trends). (B, C): The concepts shown are from gene expression data previously published and reanalyzed for the purposes of the present report [25]. Abbreviations: CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; hES, human embryonic stem (cell); Hes3, hairy and enhancer of split 3; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblasts; mIPS, mouse induced pluripotent stem (cell); SSEA1, stage-specific embryonic antigen 1; Shh, sonic hedgehog; STAT3, signal transducer and activator of transcription 3.

The therapeutic potential of manipulating the components of this pathway has been demonstrated in a series of studies showing powerful protective effects in the brain when pharmacological activators are introduced into the brain in various models of neurodegenerative disease [3, 8, 1517]. STAT3-Ser phosphorylation mediates carcinogenesis in xenotransplantation models of prostate cancer [6], suggesting that specific inhibitors directed against this site could be useful in treating certain cancer types, in particular those harboring CSC populations. Angiopoietin 2 is a powerful activator of the pathway, pointing toward new functions of angiogenic factors in tissue homeostasis, neurodegenerative disease, and cancer [17, 18]. The value of this pathway in drug discovery is also highlighted by the finding that the efficiency of a γ-secretase inhibitor as an anti-breast cancer drug was predicted by the levels of Hes3 in vivo [19]. That study, in particular has shown the disconnect between in vitro and in vivo effects, validating the idea that great care and thought is needed to appropriately model the signaling state of a cell in vitro. Overall, the STAT3-Ser/Hes3 signaling axis is operational in cell types from several different tissues and might mediate important functions in the context of a wide range of diseases.

The relevance of this pathway to harnessing stem cell technologies for therapeutic benefit is also exhibited by the findings that its activation results in much improved yields and electrophysiological properties of neurons generated from NSCs derived from induced pluripotent stem (iPS) cell sources [20]. More recently, Hes3 was implicated in the direct conversion of adult non-neural cells to the NSC state through a reprogramming method, along with other genes [21]. The STAT3 phosphorylation state is also critical to consider, because the differences between STAT3 tyrosine 705 (STAT3-Tyr) and STAT3-Ser phosphorylation requirements are clearly exhibited in embryonic stem (ES) and NSC systems. Mouse ES cells use STAT3-Tyr; thus, they are cultured in the presence of leukemia inhibitory factor (LIF). Human ES cells, however, do not rely on STAT3-Tyr to remain undifferentiated and are thus cultured in the presence of basic FGF (bFGF). In NSCs, STAT3-Tyr leads to gliogenic differentiation, but STAT3-Ser promotes survival.

In the present report, we discuss the aspects of signaling pathways that involve and/or intercept STAT3 that have been commonly assumed to be mediated by STAT3-Tyr but in reality might also be mediated by STAT3-Ser. We begin by the re-evaluating classic reprogramming paradigms (e.g., mouse embryonic fibroblasts [MEFs] to iPS). We also reanalyze the gene expression data in well-established paradigms of the differentiation of human embryonic stem cells to neurons, further supporting the operation of these pathways in cell conversion decisions. We extend this discussion by examining the conversion of acinar cells to insulin-producing cells, an exciting example of the transdifferentiation that can be induced both in vitro and in vivo. Revisiting the original studies, we reinterpret some of these assumptions and provide alternative interpretations, showing that STAT3-Ser could also be an important mediator in this process. Given the very strong impetus toward understanding the molecular mechanisms driving reprogramming and differentiation, state-of-the-art techniques that are already being applied in experimental therapies, it is essential to explore these new mechanisms of action. We do not aim to give an extensive account of the reprogramming field, which has been expertly provided elsewhere [22].

Is the STAT3-Ser/Hes3 Signaling Axis a Yet Undetected Mediator of Reprogramming?

To date, a common theme with this pathway is a role in the maintenance of the primitive state of cells capable of undergoing massive epigenetic decisions. This prompted us to ponder whether aspects of the pathway should be included in the thought process behind reprogramming. This is a very opportune time to do so, because, in addition to the many studies showing reprogramming using the classic developmental transcription factors, studies demonstrating reprogramming with soluble factors that activate distinct signaling pathways have also been reported. These pathways intercept the STAT3-Ser/Hes3 signaling axis, consistent with its involvement. Furthermore, its involvement appears restricted to particular stages of a cell’s development, with NSCs offering a great example. Pathways that regulate their self-renewal, stimulated by bFGF, and those that regulate differentiation (ciliary neurotrophic factor [CNTF]-driven astrocyte differentiation), in fact, oppose the activity of one another. This is reminiscent of what happens in the case of mouse ES cells, in which LIF-induced STAT3-Tyr activity (a stimulus necessary to maintain pluripotency in these cells) blocks FGF-induced extracellular signal-regulated kinase (Erk) activity (a differentiation signal) [23].

At the transcriptional level, recent studies have defined intermediate cell populations during mouse iPS cell generation and identified an early c-Myc/KLF4 wave, followed by a second Oct4/Sox2/KLF4 transcriptional wave required for reprogramming [24]. In addition, we have shown that the SoxB transcription factor family member SOX21 is induced by SOX2 during reprogramming and that SOX21 is required for iPS cell generation [25]. Consistent with our findings, interrogation of the data sets from Polo et al. revealed that Sox21 expression is increased as cells transition toward the iPS cell state. Examination of the genes in the STAT3-Ser/Hes3 signaling axis shows that Hes3 is at low levels in both parental MEFs and stable iPSCs but increases during initial reprogramming, peaking in early Oct4-positive cells (Fig. 1B). Hes3 abruptly decreases as the pluripotent state is locked in; interestingly, the mitogen/morphogen sonic hedgehog (Shh) shares this same expression profile. These data suggest that Hes3 is involved in the critical transition to the stable iPS state during reprogramming. The downregulation of Hes3 in the mouse pluripotent state is consistent with the dependence of mouse ES cells on the canonical JAK/STAT pathway, in contrast to other stem cell populations.

Analysis of RNAseq data sets from human ES cells [26] shows that they express Hes3 in the self-renewing state (as seen with NSCs) and display a marked increase in Hes3 expression during the first steps toward neurectodermal specification (Fig. 1C). The polycomb protein Bmi1 [2729], an important regulator of stem cell self-renewal, is also increased at this same time point and JAK1 expression is repressed. As these cells further commit and differentiate toward neural fates, Hes3 is rapidly downregulated. In cultures of fetal NSCs, Hes3 overexpression induces production of Shh, a known mitogen for NSCs [8]. Shh is also a positive modulator of BmI1, which leads to the transdifferentiation of mouse fibroblasts to NSC-like cells [30]. Taken together, these data suggest a possible Hes3-Bmi1-Shh axis that could also be involved in the reprogramming of cells to the NSC state.

Epigenetic reprogramming by genetic means has generated new sources for cell replacement strategies [22, 31, 32]. Reprogramming has also been achieved through the use of soluble factors. For example, intraperitoneal administration of a combination of epidermal growth factor (EGF) and CNTF converts acinar cells to β-like cells in vivo with consequences for regulating blood glucose in rodent models of diabetes [33]. These factors were chosen, in part, because of their broad range of functions in cell proliferation and gene regulation and because they have been implicated in various paradigms of regeneration and cell type conversion. This is of particular interest, because it provides a “handle” that is upstream of well-studied signaling pathways, providing both a molecular rationale and additional opportunities for manipulation within these signaling pathways. In a recent study, this logic led to the implementation of genetic overexpression of constitutively activated mitogen-activated protein kinase (MAPK) (caMAPK; specifically, p42/44 also known as ERK1/2) and constitutively activated STAT3 (caSTAT3) in human acinar cells in vitro, which also resulted in the reprogramming of these cells to β-like cells [34].

The simple interpretation of these data is that caMAPK is a surrogate for EGF and that caSTAT3 is a surrogate for CNTF (Fig. 2A). This might well be correct; however, a series of observations suggest the involvement of additional, noncanonical signaling pathways in this reprogramming process and open the possibility that the STAT3-Ser/Hes3 signaling axis might be involved. First, it is important to clarify that caSTAT3 models some, but not all, functions of tyrosine-phosphorylated STAT3. One of these functions is the dimerization of STAT3, which, in caSTAT3 is induced without the need for tyrosine phosphorylation. To achieve this, cysteine residues were engineered on the STAT3 monomer rendering it capable of dimerization through disulfide bonds without the need for tyrosine phosphorylation [35]. caSTAT3 can bind to DNA and activate the transcription of certain genes. However, when stimuli that lead to phosphorylation of STAT3 on tyrosine are used, such as interleukin-6 or v-src, the transcriptional activity of caSTAT3 is greatly enhanced, demonstrating the forced dimerization alone is insufficient in modeling the entire range of functions allocated to tyrosine-phosphorylated STAT3 [35, 36]. Likewise, caSTAT3, in which the tyrosine 705 residue has been mutated to phenylalanine (and cannot, therefore, be phosphorylated), fails to transactivate STAT3 target genes [37]. However, caSTAT3 was found to be serine phosphorylated, suggesting that it might be able to efficiently model the functions of this modification [35]. Therefore, the biological output after transduction with caSTAT3 cannot clearly allocate function to the phosphorylation event of one particular residue.

Figure 2.

Possible noncanonical signaling pathway involvement in reprogramming through modulators of STAT3 and MAPK. (A): Common view for the mechanism of reprogramming in acinar-to-β-cell reprogramming downstream of CNTF and EGF. STAT3-Tyr and activated MAPK induce vast transcriptional changes leading to fate specification changes. (B): Hes3 as a regulator of Ngn3 in the context of endocrine pancreas regeneration. A lack of Ngn3 expression induction in Hes3-null (Hes3−/−) mice 5 months after a low-dose streptozotocin regimen (5 consecutive daily injections at 50 mg/kg in phosphate-buffered saline [PBS]; vehicle controls received only PBS). (C): STAT3-Ser as a putative mediator of reprogramming of acinar-to-β cell conversion. Three possible alternative interpretations for the mechanism of action of CNTF- and EGF-induced reprogramming that involve the STAT3-Ser/Hes3 signaling axis. (Both CNTF and EGF lead to the phosphorylation of STAT3-Ser and STAT3-Tyr; the diagrams highlight the particular phosphorylation event that might be driving a given function. It is not meant to suggest that only one residue is phosphorylated. Also, a predominant function of STAT3-Tyr phosphorylation is the dimerization of STAT3. For this reason, and for simplicity, the diagrams depict STAT3-Tyr phosphorylation to also represent STAT3 dimerization). (B): Image width: 534 μm. Abbreviations: CNTF, ciliary neurotrophic factor; DAPI, 4′,6-diamidino-2-phenylindole; EGF, epidermal growth factor; Hes3, hairy and enhancer of split 3; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; STAT3, signal transducer and activator of transcription 3; STZ, streptozotocin.

A clue to the possible involvement of the STAT3-Ser/Hes3 signaling axis in reprogramming comes from observations of the induction of Ngn3 in pancreatic cells. These findings imply the onset of de- and transdifferentiation events, for example, in the conversion of acinar to β cells [38, 39]. Specifically, transduction with caMAPK and caSTAT3 induced Ngn3 only in acinar cells and not in β cells, although both cell types express Ngn3 [34]. In accordance with the involvement of the components of the STAT3-Ser/Hes3 signaling axis in Ngn3 induction in these different cell populations, Hes3-null mice fail to induce Ngn3 in the regenerating pancreas after streptozotocin (STZ) damage. Wild-type mice exhibit Ngn3 expression in both pancreatic islet β cells and acinar cells. At 5 months after STZ-induced damage, Ngn3 expression in acinar cells is strongly upregulated, with no detectable changes in β cells (Fig. 2B). Like wild-type mice, nondamaged Hes3-null mice also exhibit Ngn3 expression in both β and acinar cells. However, after STZ damage, the upregulation of Ngn3 is greatly attenuated. These results suggest the involvement of Hes3 in the induction of Ngn3 in acinar cells and therefore support the hypothesis that caMAPK and caSTAT3 operate through the STAT3-Ser/Hes3 signaling axis in this context.

Another clue is that the order of viral transduction matters. For reprogramming to work efficiently, caMAPK must be transduced before caSTAT3. Transduction of both constructs simultaneously resulted in lower efficiency. Transduction of caSTAT3 before caMAPK resulted in even lower efficiency. Each gene alone was insufficient for reprogramming. We present three alternative interpretations of these findings (Fig. 2C).


Alternative Interpretation 1: STAT3-Ser-Driven Reprogramming

It is possible that, in part, the effects of this reprogramming method involve STAT3-Ser phosphorylation. This would explain why the order of caMAPK first and caSTAT3 second is so important in the efficiency of reprogramming. caMAPK leads to powerful STAT3-Ser phosphorylation via several pathways [40]. Therefore, an initial overexpression of caMAPK would ensure that a subsequent overexpression of caSTAT3 would immediately result in high amounts of STAT3 that would be dimerized and serine phosphorylated. In contrast, the reverse order would result in high amounts of STAT3 that would be dimerized, with possibly submaximal serine phosphorylation, leading to potentially different gene regulation. It is also possible that caMAPK might contribute to reprogramming by elevating serine phosphorylation of endogenous STAT3. This could be tested with experiments in which serine-phosphomimetic constructs of STAT3 that cannot be tyrosine phosphorylated can be assessed for their potential to contribute to reprogramming.


Alternative Interpretation 2: JAK Titration

Although JAK activity is predominantly seen as a means of elevating STAT3-Tyr phosphorylation, JAK also leads to STAT3-Ser phosphorylation [40]. The distinction between these two phosphorylation events can be regulated by the levels of cytokine activity that lead to JAK activation [8]. For example, low levels of CNTF in NSC cultures result in the elevation of STAT3-Ser phosphorylation with no indication of an effect on STAT3-Tyr phosphorylation. Higher CNTF concentrations lead to abrupt increases in STAT3-Tyr phosphorylation [5]. Therefore, CNTF levels can be used either to promote STAT3-Ser phosphorylation in the absence of detectable STAT3-Tyr phosphorylation (and, therefore, promote NSC self-renewal and increase cell numbers) or to promote both serine and tyrosine phosphorylation (and, therefore, inhibit self-renewal and induce differentiation). In another example, the use of a JAK inhibitor in GBM CSCs can significantly increase the ratio of STAT3-Ser to STAT3-Tyr phosphorylation, and as a consequence, the expression of Hes3. This leads to changes in various properties of these cells, including the ability to achieve higher terminal cell densities in vitro [5]. In this scenario, specific levels of JAK activity—which can be regulated using a JAK inhibitor [34, 41]—might result in the stimulation of STAT3-Ser, in the absence of STAT3-Tyr, and promote reprogramming. Therefore, although the tyrosine site might be very important in this reprogramming paradigm, it is also possible that the serine site is also an important player in reprogramming.


Alternative Interpretation 3: A Two-Step Process

It is also possible that the serine and tyrosine phosphorylation of STAT3 represent two distinct events within the reprogramming process and that both are necessary for reprogramming. It is conceivable that the serine event must precede the tyrosine event, as suggested by the observations regarding the importance of order for caMAPK and caSTAT3.

STAT3-Ser/Hes3 in Cancer—Clues to a Role in Reprogramming

Key reprogramming factors are oncogenes, highlighting the similarities between developmental programs and transformation. Hes3 might belong to this category. Direct evidence for this comes from work showing that Hes3 RNA interference opposes the growth of putative CSCs from glioblastoma multiforme biopsies [5], and supporting its putative role in breast cancer, where in vivo Hes3 expression levels correlated with the efficacy of a γ-secretase inhibitor [19]. Other key components of the STAT3-Ser/Hes3 signaling axis are also implicated in carcinogenesis. For example, STAT3-Ser phosphorylation in the absence of STAT3-Tyr phosphorylation has also been demonstrated to drive prostate carcinogenesis [6]. Therefore, the STAT3-Ser/Hes3 signaling axis might represent an oncogenic, noncanonical branch of the Notch signaling pathway, providing an explanation of the dual properties of Notch signaling as both an oncogene and a tumor suppressor [42, 43].

In line with a dynamic role for Hes3 in regulating the epigenetic state of a cell, Hes3 expression and subcellular localization are themselves dynamically and, in certain cases, reversibly, regulated. Hes3 expression and subcellular localization can also provide clues to its function and reveal information of the state of a cell. GBM CSCs and a mouse insulinoma cell line (MIN6) can be efficiently cultured under conditions that support nuclear Hes3 expression or prevent it [5, 7]. By changing the culture conditions, the cells can be repeatedly switched from one state to the other. For example, serum-free defined cultured conditions allow the nuclear expression of Hes3. Under these conditions, the cells grow independently of JAK activity. In fact, inclusion of a JAK inhibitor promotes GBM CSC growth. Primary, bona fide NSC cultures also exhibit Hes3 expression, which is lost when the cells are induced to differentiate, similar to GBM CSCs. In contrast, however, primary NSCs do not exhibit reversibility in their differentiation and Hes3 expression states, a property that has been proposed to characterize the CSC population [44]. It could be of use in assays determining the NSC state to include measurements of the irreversibility of the differentiation of these cells.


The STAT3-Ser/Hes3 signaling axis has been implicated in a number of phenomena, including improved culture conditions for plastic cell types, their activation in vivo in the context of neurodegenerative disease, as a target for brain CSCs, and as a mediator of pancreatic β-cell function and survival. More recently, the pathway has also been implicated in aspects of reprogramming to the NSC state. An understanding of this signaling pathway will provide possible molecular explanations for its function in the context of reprogramming. The possibility that Hes3 could prove to be an important player in different aspects of epigenetic reprogramming as manifested in the iPS and CSC fields warrants more investigation.

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What’s new with CRISPR-Cas9?

Larry H. Bernstein, MD, FCAP, Curator


Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

Author: Larry H. Bernstein, MD, FCAP


2.2.18   CRISPR-Cas9 and Regenerative Medicine, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair


There has been a rapid development of methods for genetic engineering that is based on an initial work on bacterial resistance to viral invasion.  The engineering called RNA inhibition (RNAi) has gone through several stages leading to a more rapid and more specific application with minimal error.

It is a different issue to consider this application with respect to bacterial, viral, fungal, or parasitic invasion than it would be for complex human metabolic conditions and human cancer. The difference is that humans and multi-organ species are well differentiated systems with organ specific genome translation to function.

I would expect to see the use of genomic alteration as most promising in the near term for the enormous battle against antimicrobial, antifungal, and antiparasitic drug resistance.  This could well be expected to be a long-term battle because of the invading organisms innate propensity to develop resistance.

A CRISPR/Cas system mediates bacterial innate immune evasion and virulence

Timothy R. Sampson, Sunil D. Saroj, Anna C. Llewellyn, Yih-Ling Tzeng David S. Weiss

Affiliations, Contributions, Corresponding author

Nature 497, 254–257 (09 May 2013),  http://dx.doi.org:/10.1038/nature12048

CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids1234. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation5678. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes910. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens1112, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.




Zhang lab unlocks crystal structure of new CRISPR/Cas9 genome editing tool

Paul Goldsmith,  2015 Aug

In a paper published today in Cell researchers from the Broad Institute and University of Tokyo revealed the crystal structure of theStaphylococcus aureus Cas9 complex (SaCas9)—a highly efficient enzyme that overcomes one of the primary challenges to in vivo mammalian genome editing.

First identified as a potential genome-editing tool by Broad Institute core member Feng Zhang and his colleagues (and published by Zhang lab in April 2015), SaCas9 is expected to expand scientists’ ability to edit genomes in vivo. This new structural study will help researchers refine and further engineer this promising tool to accelerate genomic research and bring the technology closer to use in the treatment of human genetic disease.

“SaCas9 is the latest addition to our Cas9 toolbox, and the crystal shows us its blueprint,” said co-senior author Feng Zhang, who in addition to his Broad role, is also an investigator at the McGovern Institute for Brain Research, and an assistant professor at MIT.

The engineered CRISPR-Cas9 system adapts a naturally-occurring system that bacteria use as a defense mechanism against viral infection. The Zhang lab first harnessed this system as an effective genome-editing tool in mammalian cells using the Cas9 enzymes from Streptococcus thermophilus (StCas9) andStreptococcus pyogenes (SpCas9). Now, Zhang and colleagues have detailed the molecular structure of SaCas9, providing scientists with a high-resolution map of this enzyme. By comparing the crystal structure of SaCas9 to the crystal structure of the more commonly-used SpCas9 (published by the Zhang lab in February 2014), the team was able to focus on aspects important to Cas9 function— potentially paving the way to further develop the experimental and therapeutic potential of the CRISPR-Cas9 system.

Paper cited: Nishimasu H et al. “Crystal Structure of Staphylococcus aureus Cas9.” Cell, http://dx.doi.org:/10.1016/j.cell.2015.08.007

Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

Rodolphe Barrangou1,†, Amanda Birmingham2,†, Stefan Wiemann3, Roderick L. Beijersbergen4, Veit Hornung5 and Anja van Brabant Smith2
Nucleic Acids Research, 2015 Mar 23.  http:dx.doi.org:/10.1093/nar/gkv226

RNAi and CRISPR-Cas9 have many clear similarities. Indeed, the mechanisms of both use small RNAs with an on-target specificity of ∼18–20 nt. Both methods have been extensively reviewed recently (3–5) so we only highlight their main features here. RNAi operates by piggybacking on the endogenous eukaryotic pathway for microRNA-based gene regulation (Figure 1A). microRNAs (miRNAs) are small, ∼22-nt-long molecules that cause cleavage, degradation and/or translational repression of RNAs with adequate complementarity to them(6).RNAi reagentsfor research aim to exploit the cleavage pathway using perfect complementarity to their targets to produce robust downregulation of only the intended target gene. The CRISPRCas9 system, on the other hand, originates from the bacterial CRISPR-Cas system, which provides adaptive immunity against invading genetic elements (7). Generally, CRISPR-Cas systems provide DNA-encoded (7), RNAmediated (8), DNA- (9) or RNA-targeting(10) sequencespecific targeting. Cas9 is the signature protein for Type II CRISPR-Cas systems (11).


Both RNAi and CRISPR-Cas9 have experienced significant milestones in their technological development, as highlighted in Figure 2 (7–14,16–22,24–51) (highlighted topics have been detailed in recent reviews (2,4,52–58)). The CRISPR-Cas9 milestones to date have mimicked a compressed version of those for RNAi, underlining the practical benefit of leveraging similarities to this well-trodden research path. While RNAi has already influenced many advances in the CRISPR-Cas9 field, other applications of CRISPR-Cas9 have not yet been attained but will likely continue to be inspired by the corresponding advances in the RNAi field (Table 1). Of particular interest are the potential parallels in efficiency, specificity, screening and in vivo/therapeutic applications, which we discuss further below.

Figure2. Timeline of milestones for RNAi and CRISPR-Cas9. Milestones in the RNAi field are noted above the line and milestones in the CRISPR-Cas9 field are noted below the line. These milestones have been covered in depth in recent reviews (2,4,52–29).
Table 1. Summary of improvements in the CRISPR-Cas9 field that can be anticipated by corresponding RNAi advances

more….  see at  http://pharmaceuticalintelligence.com/2015/09/01/where-is-the-most-promising-avenue-to-success-in-pharmaceuticals-with-crispr-cas9/

Early Diagnosis


Reporter: Stephen J. Williams, Ph.D.

This post contains a curation of all Early Diagnosis posts on this site as well as a curation of the Early Detection Research Network.

Highlights of the accomplishments of the Early Detection Research Network.

A brief list of major EDRN-developed assays that have been adapted for clinical use is described in the table below:

Detection/Biomarker Assay Discovery Refine/Adapt for Clin Use Clinical Validation Clinical Translation
Blood proPSA FDA approved
Urine PCA3 FDA approved
OVA1™ for Ovarian Cancer FDA approved
ROMA Algorithm for CA125 and HE4 Tests for Pelvic Mass Malignancies FDA approved
Blood/DCP and AFP-L3 for Hepatocellular Carcinoma FDA approved
Blood GP73 Together with AFP-L3 used  for monitoring cirrhotic patients for HCC in China
MiPS (Mi Prostate Score Urine test), Multiplex analysis of T2-ERG gene fusion, PCA3 and serum PSA In CLIA Lab
FISH to detect T2S:Erg fusion for Prostate Cancer In CLIA Lab
GSTP1 methylation for repeat biopsies in prostate cancer In CLIA Lab
Mitochondrial deletion for detection of prostate cancer In CLIA Lab
Somalogic 12-marker panel for Lung Cancer In CLIA Lab
80-gene panel for Lung Cancer In CLIA Lab
Vimentin Methylation Marker for Colon Cancer In CLIA Lab
Galectin-3 ligand for detection of adenomas and colon cancer In CLIA Lab
8-gene panel for Barrett’s Esophagus In CLIA Lab
SOPs for Blood (Serum, Plasma), Urine, Stool Frequently used by biomarker research community
EDRN Pre/Validation Specimen Reference Sets (specimens from well characterized and matched cases and controls from specific disease spectra) Frequently used by biomarker research community

Since its inception in 1999 EDRN has achieved several key milestones, summarized below:

1998 through 2000: Inception and Inauguration of EDRN


The European Society for Gene and Cell Therapy and the Spanish Society for Gene and Cell Therapy Collaborative Congress 2013

HUMAN GENE THERAPY XX:A2–A172 (XXXX 2013) ª Mary Ann Liebert, Inc.   http://dx.doi.org:/10.1089/hum.2013.2513

Bases of gene therapy in leukemias
C. Bonini Experimental Hematology Unit, Division of Regenerative Medicine, Gene Therapy and Stem Cells,
Program of Immunology, Gene Therapy and Bio-Immunotherapy of Cancer, Leukemia Unit, San Raffaele Scientific Institute, Milan, Italy

Hematopoietic stem cell transplantation from a healthy donor (allo-HSCT) represents the most potent form of cellular adoptive immunotherapy to treat leukemias. During the past decades, allo-HSCT has developed from being an experimental therapy offered to patients with end-stage leukemia into a wellestablished therapeutic option for patients affected by several hematological malignancies. In allo-HSCT, donor T cells are double edge-swords, highly potent against residual tumor cells, but potentially highly toxic, and responsible of the graft versus host disease (GVHD), a major clinical complication of transplantation. Gene transfer technologies can improve the safety (ie: use of suicide genes), and the efficacy (ie: TCR gene transfer, TCR gene editing, CAR gene transfer) of adoptive T-cell therapy in the context of allo-HSCT. The encouraging preclinical and clinical results obtained in these years with genetically engineered T lymphocytes in the treatment of leukemias will be discussed.

Recent developments in gene therapy of solid tumors
R. Hernandez Division of Gene Therapy and Hepatology,
Universidad de Navarra, Madrid, Spain

Treatment of cancer has been one of the earliest and most frequent applications of gene therapy in experimental medicine. However, this indication entails unique difficulties, especially in the case of solid tumors. Pioneering strategies were aimed to reverse the malignant phenotype or to induce the death of cancer cells by transferring tumor-suppressor genes, inhibiting oncogenes or selectively expressing toxic genes. Proof of principle has been generated in abundant pre-clinical models and in humans. However, clinical efficacy is hampered by the diffi- culty in delivering therapeutic genes to a significant proportion of cancer cells in solid tumors using the currently available vectors. Therefore, current work aims to extend the effect to non-transduced cancer cells. This can be achieved by local or systemic expression of secreted proteins with the ability to block key pathways involved in angiogenesis, cell proliferation and invasion. Recent advances in gene therapy vectors allow sustained expression of transgenes and make these strategies feasible in the clinic. Another attractive option is the stimulation of immune reactions against cancer cells using gene transfer. In this case the therapeutic genes are antigens, cytokines or proteins capable of blocking the immunosuppressive microenvironment of tumors. Adaptation of replication-competent (oncolytic) viruses as vectors for these genes combines the intrinsic immunogenicity of viruses, their capacity to amplify gene expression and their direct lytic effect on cancer cells. In general, the ‘‘immunogene therapy’’ strategies offer the opportunity to destroy primary and distant lesions, especially if they are combined with other treatments that reduce tumor burden. More importantly, vaccination against cancer cells could prevent cancer relapse. Finally, gene and cell therapies are joining forces to improve the efficacy of adoptive cell therapy. Ex vivo gene transfer of natural or chimeric tumor-specific receptors in T lymphocytes enhances the cytotoxic potency of the cells and is expanding the applicability of this promising approach to different tumor types.

Production of vector and genetically modified stem cells
A. Galy and E. de Barbeyrac Genethon, 1
bis rue de l’Internationale, F91002 Evry, France

Hematopoietic gene therapy is currently used to treat a variety of genetic disorders of the blood and immune systems, or metabolic diseases, with promising results. The approach currently relies on the infusion of patient-autologous hematopoietic stem cells that have been subjected to gene-transfer ex vivo with a viral vector of clinical grade, during a short period of culture. The manufacture of such advanced therapy medicinal products for clinical trials should comply with the clinical trials EC directive. Requirements for gene and cell-based medicinal products both apply, therefore a high level of complexity is involved in the development of such products. Hematopoietic cell and gene therapy has many potential indications based on encouraging preclinical and early-phase clinical results. However, somatic cell and gene therapy medicinal products are still in early phases of development and no such product has been registered yet. The standardization of the manufacturing process and characterization of the drug product (i.e. geneticallymodified cells) are important but present challenges. Many aspects, and in particular limited available patient material, complicate a precise characterization of the drug product. On the other hand, clinical-grade gene transfer retroviral vectors are well-characterized starting materials that are described in a pharmacopeia monograph and can be robustly manufactured in successive campaigns of production under GMP conditions. Examples obtained in preclinical and ongoing clinical studies to treat Wiskott Aldrich Syndrome illustrate the vast differences in the level of characterization between the viral vector starting material and the drug product used in hematopoietic gene therapy. Characterization of the products and standardization/ validation of the manufacturing process are the next challenges in the field.

Gammaretro and lentiviral vectors for the gene therapy of X-linked chronic Granulomatous disease
M. Grez Institute for Biomedical Research,
Georg-Speyer-Haus, Frankfurt, Germany

Gene therapy of inherited diseases has provided convincing evidence of therapeutic benefits for many treated patients. In particular, treatment of primary severe congenital immunodeficiencies by gene transfer into hematopoietic stem cells (HSCs) has proven in some cases to be as beneficial as allogeneic stem cell transplantation, the treatment of choice for these diseases if HLA-matched donors are available. We conducted a Phase I clinical trial aimed at the correction of X-CGD, a rare inherited immunodeficiency characterized by severe and life threatening bacterial and fungal infections as well as widespread tissue granuloma formation. Phagocytic cells of CGD patients fail to kill ingested microbes due to a defect in the nicotinamide dinucleotide phosphate (NADPH) oxidase complex resulting in compromised antimicrobial activity. In this clinical trial we used a gammaretroviral vector with strong enhancer-promoter sequences in the long terminal repeats (LTRs) to genetically modify CD34 + cells in two X-CGD patients. After successful reconstitution of phagocytic functions, both patients experienced a clonal outgrowth of gene marked cells caused by vector-mediated insertional activation of proto-oncogenes leading to the development of myeloid malignancies. Moreover, functional correction of gene transduced cells decreased with time, due to epigenetic inactivation of the vector promoter within the LTR, resulting in the accumulation of nonfunctional gene transduced cells. The understanding of the molecular basis of insertional mutagenesis has motivated the development of advanced integrating vectors with equal therapeutic potency but reduced genotoxicity. In particular, the deletion of the enhancer elements within the viral LTR U3 regions has significantly contributed to the reduction of genotoxic effects associated with LTR-driven gammaretroviral vectors. Moreover, the use of tissue specific promoters, which are inactive in stem/progenitor cells but active in terminally differentiated cells, should further increase the safety level of SIN vectors. Based on the aforementioned advancements, we developed SIN gammaretroviral and lentiviral vectors for the safe and effective gene therapy of X-linked CGD. We combined the SIN configuration with an internal promoter, with preferential expression in myeloid cells. However, the introduction of a new vector into the clinic demands a series of sophisticated pre-clinical studies, which are quite challenging in particular within an academic environment. In this presentation we will report on the comprehensive and thorough preclinical efficacy and safety testing of both SIN vectors assessing dosage requirements, therapeutic efficacy, resistance to transgene silencing and genotoxic potential.

Progress and challenges of in vivo gene transfer with AAV vectors
F. Mingozzi1,2 1 Genethon, Evry, France; 2
University Pierre and Marie Curie, Paris, France

In vivo gene replacement for the treatment of an inherited disease is one of the most compelling concepts in modern medicine. Adeno-associated virus (AAV) vectors have been extensively used for this purpose and have shown therapeutic efficacy in a range of animal models. The translation of preclinical results to the clinic was initially slow, but early studies in humans helped defining the roadblocks to successful therapeutic gene transfer in vivo, which are highly depending on the target tissue, the route of vector delivery, and the specific disease. The development of strategies to overcome these limitations allowed achieving long-term expression of donated genes at therapeutic levels in patients with inherited retinal disorders, hemophilia B and other diseases. The recent market approval of Glybera, an AAV vector-based gene therapy product for lipoprotein lipase deficiency, further con- firmed the potential of AAV vectors as a therapeutic platform, raising hopes for the development of in vivo gene transfer treatments for many additional inherited and acquired diseases.

Glybera approval: a road map for advanced therapies in the orphan space
H. Petry
uniQure, Amsterdam, Netherlands

Glybera, is a gene therapy product based on the use of recombinant adeno-associated virus for gene delivery. It is designed for patients with Lipoprotein Lipase Deficiency (LPLD). On November 2, 2012, the European Commission approved the marketing authorisation for Glybera as a treatment for LPLD, under exceptional circumstances, in all 27 EU member states. Glybera is intended to treat patients with lipoprotein lipase deficiency. LPLD is caused by errors in the gene that codes for the protein lipoprotein lipase (LPL). LPL has a central role in fat metabolism. Non-functional LPL can lead to pancreatitis attacks, the most sever phenotype of this disease. The presentation will cover a summary of the clinical development, as well as a summary of the regulatory process. In addition post approval commitments will be discussed and their importance to follow up on the long term safety and efficacy of the this gene therapy product.

Phase Ib/IIa, escalating dose, single blind, clinical trial to assess the safety of the intravenous administration of expanded allogeneic adipose-derived mesenchymal stem cells (eASCs) to refractory rheumatoid arthritis (RA) patients
L. Dorrego
Tigenix, Madrid, Spain

Advanced therapies are emerging and fast-growing biotechnology sector paves the way for new, highly promising treatment opportunities for European patients. TiGenix is a leading European cell therapy company a marketed product for cartilage repair, and a strong pipeline with advanced clinical stage allogeneic adult stem cell programs for the treatment of autoimmune and inflammatory diseases. TiGenix has developed an innovative trial design in the stem cell area for treating refractory rheumatoid arthritis (RA) using expanded allogeneic adipose-derived mesenchymal stem cells (eASCS). The multicenter, randomized, double blind, placebocontrolled Phase IIa trial enrolled 53 patients with active refractory rheumatoid arthritis (mean time since diagnosis 15 years), who failed to respond to at least two biologics (mean previous treatment with 3 or more disease-modifying antirheumatic drugs and 3 or more biologics). The study design was based on a threecohort dose-escalating protocol. For both the low and medium dose regimens 20 patients received active treatment versus 3 patients on placebo; for the high dose regimen 6 patients received active treatment versus 1 on placebo. Patients were dosed at day 1, 8, and 15 and were followed up monthly over a six-month period. Follow-up consisted of a detailed monthly workup of all patients measuring all pre-defined parameters. The aim was to evaluate the safety, tolerability and optimal dosing over the full 6 months of the trial, as well as exploring therapeutic activity. Twenty five Spanish sites participated in this clinical trial. Coordinating Investigator: Dr. Jose´ Marı´a Alvaro-Gracia

Induction of multi-, pluri- and totipotency
H.R. Scho¨ler
Department Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Muenster, 48149, Germany

The pluripotent and multipotent states of stem cells are governed by the expression of few, specific transcription factors forming a highly interconnected regulatory network with more numerous, widely expressed transcription factors. When the set of master transcription factors comprising Oct4, Sox2, Klf4, and Myc is expressed ectopically in somatic cells, this network organizes itself to support a pluripotent cell state. But when Oct4 is replaced by Brn4, another POU transcription factor, fibroblasts are converted into multipotent neural stem cells. These two transcription factors appear to play distinct but interdependent roles in remodelling gene expression by influencing the local chromatin status during reprogramming. Furthermore, structural analysis of Oct4 bound to DNA shows that the Oct4 linker—a region connecting the two POU domains of Oct4—is exposed to the surface, and we therefore postulate that it recruits key epigenetic players onto Oct4 target genes during reprogramming. The role of Oct4 in defining totipotency and inducing pluripotency during embryonic development remains unclear, however. We genetically eliminated maternal Oct4 using a Cre/ lox approach and found no effect on the establishment of totipotency, as shown by the generation of live pups. After complete inactivation of both maternal and zygotic Oct4 expression, the embryos still formed Oct4-GFP– and Nanog–expressing inner cell masses, albeit nonpluripotent, indicating that Oct4 is not a determinant for the pluripotent cell lineage separation. Interestingly, Oct4-deficient oocytes were able to reprogram fibroblasts into pluripotent cells. Our results indicate that, in contrast to its crucial role in the maintenance of pluripotency, maternal Oct4 is crucial for neither the establishment of totipotency in embryos, nor the induction of pluripotency in somatic cells using oocytes.

Reprogramming in vivo is possible and generates a new type of iPS
M. Serrano
Spanish National Cancer Research Center (CNIO), Madrid, Spain

Reprogramming into induced pluripotent stem cells (iPSCs) has opened new therapeutic opportunities, however, little is known about the possibility of in vivo reprogramming within tissues. We have generated transgenic mice with inducible expression of the four Yamanaka factors. Interestingly, transitory induction of the reprogramming factors results in teratomas emerging from multiple organs, thereby, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. Also, by bone marrow transplantation, we demonstrate that hematopoietic cells can also be reprogrammed in vivo. Remarkably, induced reprogrammable mice also present circulating iPSCs in the blood. These in vivo-generated iPSCs can be purified and grown (in the absence of further induction of the reprogramming factors). Strikingly, at the transcriptome level, the in vivo-generated iPSCs are closer to embryonic stem cells (ESCs) than to standard in vitro-generated iPSCs. Moreover, in vivo-iPSCs efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ESCs. Finally, in vivo-iPSCs show an unprecedented capacity to form embryo-like structures upon intraperitoneal injection, including the three germ layers of the proper embryo and extraembryonic tissues, such as extraembryonic ectoderm and yolk sac-like with associated embryonic erythropoiesis. These capacities are absent in ESCs or in standard in vitro-iPSCs. In summary, in vivo-iPSCs represent a more primitive or plastic state than ESCs or in vitro-iPSCs. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

Sleeping Beauty transpsons for molecular medicine
J.C. Izpisua
Belmonte Salk Institute for Biological Studies, La Jolla, CA, USA

The development of gene-editing technologies in combination with the generation of patient-specific induced pluripotent stem cells (iPSCs) represents the merge of both the stem cell and gene therapy fields. Novel gene-editing technologies in combination with iPSCs derivation methodologies open the possibility not only for direct gene therapy but also for the replenishment of loss and/or defective cell populations with gene-corrected cells. We will present recent examples developed in our laboratory to illustrate some of the different approaches being undertaken in these fields.

The Sleeping Beauty transposon system for molecular medicine
Z. Ivics
Paul Ehrlich Institute, Langen, Germany

Non-viral gene transfer approaches typically result in only short-lived transgene expression in primary cells, due to the lack of nuclear maintenance of the vector over time and cell division. The development of efficient and safe non-viral vectors armed with an integrating feature would thus greatly facilitate clinical gene therapy studies. The latest generation transposon technology based on the Sleeping Beauty (SB) transposon may potentially overcome some of these limitations. SB was recently shown to provide efficient stable gene transfer and sustained transgene expression in primary cell types, including human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), iPSCs and T cells. The first-in-man clinical trial has been launched to use redirected T cells engineered with SB for gene therapy of B cell lymphoma. In addition, an EU FP7 project was recently initiated with the aim of replacing degenerated retinal pigment epithelial cells with cells that have been genetically modified by SB gene vectors ex vivo to produce an anti-angiogenic and neuroprotective factor for the potential treatment of patients suffering from age-related macular degeneration.

X-reactivation impacts human iPSC differentiation potential towards blood
N-B. Woods
Lund’s Stem Cell Center, Lund University, Sweden

To determine novel key regulators that direct ES/iPS cell differentiation to hematopoietic lineages, we compared the gene expression profiles of multiple iPS cell lines with differential blood forming capacity. We generated multiple iPS cell lines from amniotic fluid derived mesenchymal stromal cells (AFiPS) which differentiated towards hematopoietic lineages using our standardized and highly reproducible differentiation protocol. Of the 9 AF-iPS cell lines derived from an individual female patient, the average efficiency of CD45 + hematopoietic cells was 14.2 + / – 9% (range 1.6 to 26.3%). To elucidate the possible reasons for this diversity in efficiency, we grouped the AF-iPS cell lines on the basis of lowest and highest blood differentiation capacity and compared their gene expression pro- files by microarray. We found very few changes above 1.5-fold, but interestingly, among the 11 genes that were over-expressed in the AF-iPSC lines with poor blood differentiation efficiency, 10 were located on X chromosome, and the remaining one reported to be involved in Notch signalling. A combination of cumulative sum analysis and the location of differentially expressed genes on the X chromosome identified putative regions of reactivation at multiple, but distinct locations. The possibility of X-reactivation in these female lines was reinforced further where lower levels of XIST were seen in AF-iPSC lines shown to have low blood forming potential, however only half of the iPS cell lines with high blood differentiation capacity showed normal XIST expression when compared to the amniotic fluid mesenchymal starting cell material. To determine whether the block in differentiation was tissue specific we tested the differentiation capacity of the AF-iPSC lines towards neuronal lineages. Intriguingly, we found neural cell differentiation was not hampered within all lines with poor blood potential suggesting that the over-expression of genes as a consequence of X-reactivation can impart a specific negative effect on differentiation towards the blood lineages from pluripotency stage, while not having an effect on neuronal cell development. To further define the source of this block, we have begun working knocking down the overexpressed genes on X chromosome in lines with poor blood differentiation potential to determine whether the efficiency can be increased (or fully rescued) with one, or a combination of these 11 candidate genes. These results have implications for the identification and selection of female iPS lines suitable for therapeutic purposes. I will also discuss the identification of three new factors for improving blood lineage potential of iPS cells lines.

DLL4/Notch1 signaling is required for endothelial-tohematopoietic transition in a hESC model of human embryonic hematopoiesis
V. Ayllon1 , V. Ramos-Mejı´a1 , P.J. Real1 , O. Navarro-Montero1 , T. Romero1 , C. Bueno1,2, P. Menendez1,2,3 1
GENyO, Centre for Genomics & Oncological Research: Pfizer/ University of Granada / Andalusian Government, Granada, Spain; 2 Josep Carreras Leukemia Research Institute and Cell Therapy Program of University of Barcelona, Barcelona, Spain; 3 ICREA: Institucio´ Catalana de Reserca i Estudis Avanc¸ats, Catalunya Government, Spain

Notch signaling is essential for definitive embryonic hematopoiesis, but little is known on how Notch regulates hematopoiesis in early human embryonic development. Here we analyzed the contribution of Notch signaling to human embryonic hematopoietic differentiation using hESCs. We determined the expression of Notch receptors and ligands during hematopoietic differentiation of hESCs and found that expression of the Notch ligand DLL4 strongly parallels the emergence of bipotent hematoendothelial progenitors (HEPs). Co-cultures of hESCs with OP9-DLL4 cells demonstrated that DLL4 has a dual role in hematopoietic differentiation: during HEPs specification untimely DLL4-mediated Notch activation is detrimental for HEPs generation; however, once HEPs are specified, activation of Notch by DLL4 enhances hematopoietic commitment of these HEPs. We determined by flow cytometry that in hESCs differentiation, DLL4 is only expressed in a subpopulation of HEPs. Gene expression profiling of DLL4high and DLL4low/- HEPs showed that these two subpopulations already exhibit a distinct transcriptome program which determines their differentiation commitment: DLL4high HEPs are highly enriched in endothelial genes, while DLL4low/- HEPs display a clear hematopoietic transcriptional signature. Single cell cloning analysis of these two populations confirmed that DLL4high HEPs are enriched in committed endothelial precursors, while DLL4low/- HEPs contain committed hematopoietic progenitors. Confocal microscopy analysis of whole embryoid bodies revealed that DLL4high HEPs are located in close proximity to DLL4low/- HEPs, and at the base of clusters of CD45 + cells forming structures that resemble AGM hematopoietic clusters found in mouse embryos. Moreover, we found active Notch1 in clusters of emerging CD45 + cells. Overall, our data indicate that DLL4 regulates blood formation from hESCs, with DLL4high HEPs enriched in endothelial potential, whereas DLL4low/- HEPs are transcriptional and functionally committed to hematopoietic development. We propose a model for human embryonic hematopoiesis in which DLL4low/- HEPs receive a signal from DLL4high HEPs to activate Notch1, to undergo an endothelial-to-hematopoietic transition and differentiate into CD45 + hematopoietic cells, resembling what occurs in mouse AGM hematopoietic clusters.

Researchers Investigate Importance of STAT1 Phosphorylation in NK Cells

“If we can stop CDK8 from inactivating STAT1 in NK cells, we could stimulate tumor surveillance and thus possibly have a new handle on treating cancer, harnessing the body’s own weapons against malignant cells.” –Dr. Eva Maria Putz.



Mammals contain cells whose primary function is to kill other cells in the body. The so-called Natural Killer (NK) cells are highly important in defending our bodies against viruses or even cancer. Scientists at the University of Veterinary Medicine, Vienna (Vetmeduni Vienna) provide evidence that NK cell activity can be influenced by phosphorylating a protein (STAT1) in NK cells. The results, which could be of immediate therapeutic relevance, were recently published.

Since its discovery in the early 1990s, the protein STAT1 (Signal Transducer and Activator of Transcription 1) has been found to be central in passing signals across immune cells, ensuring that our bodies react quickly and appropriately to threats from viruses or other pathogens. Animals without STAT1 are also prone to develop cancer, suggesting that STAT1 is somehow involved in protection against malignant cells. The STAT1 protein is known to be phosphorylated on at least two positions: phosphorylation of a particular tyrosine (tyr-701) is required for the protein to enter the cell nucleus (where it exerts its effects), while subsequent phosphorylation of a serine residue alters the way it interacts with other proteins, thereby affecting its function.

Natural Killer (NK) cells are among the first cells to respond to infections by viruses or to attack malignant cells when tumors develop. When they detect cells to be targeted, they produce a number of proteins, such as granzyme B and perforin, which enter infected cells and destroy them from within. Clearly, the lethal activity must be tightly controlled to prevent NK cells from running wild and destroying healthy cells or tissues. How is this done?

Eva Maria Putz and colleagues at the Institute of Pharmacology and Toxicology of the University of Veterinary Medicine, Vienna (Vetmeduni) have now investigated the importance of STAT1 phosphorylation in NK cells. The researchers found that when a particular serine residue (ser-727) in the STAT1 protein is mutated, NK cells produce far higher amounts of granzyme B and perforin and are far more effective at killing a wide range of tumor cells. Mice with the correspondingly mutated Stat1 gene are far less likely to develop melanoma, leukemia, or metastasizing breast cancer. On the other hand, when the same serine residue is phosphorylated, the NK cells are less able to kill infected or cancerous cells.

The Vetmeduni researchers have accumulated a body of evidence to suggest that the cyclin-dependent kinase CDK8 phosphorylates STAT1 on serine 727. Surprisingly, this phosphorylation does not require prior phosphorylation of the activating tyrosine residue, at least in NK cells. Instead, it seems to represent a way in which the lethal activity of the NK cells is kept in check. Putz is keen to note the potential significance of the finding. As she says, “If we can stop CDK8 from inactivating STAT1 in NK cells, we could stimulate tumor surveillance and thus possibly have a new handle on treating cancer, harnessing the body’s own weapons against malignant cells.”

Illustration: Inhibition of NK cells by phosphorylation of STAT1-Serin 727 mediated by CDK8. –Eva-Maria Putz/Vetmeduni Vienna.

Read more…

University of Veterinary Medicine, Vienna News Release (09/06/13)

Important Step in Development of Artificial Nerves via Regenerative Medicine  

The new cells successfully regenerated axons and extended their growth farther across nerve cell gaps toward damaged nerve stumps, with healthier vascularity.



A study carried out by researchers at the Kyoto University School of Medicine has shown that when transplanted bone marrow cells (BMCs) containing adult stem cells are protected by a 15mm silicon tube and nourished with bio-engineered materials, they successfully help regenerate damaged nerves. The research may provide an important step in developing artificial nerves.

“We focused on the vascular and neurochemical environment within the tube,” said Tomoyuki Yamakawa, MD, the study’s lead author. “We thought that BMCs containing adult stem cells, with the potential to differentiate into bone, cartilage, fat, muscle, or neuronal cells, could survive by obtaining oxygen and nutrients, with the result that rates of cell differentiation and regeneration would improve.”

Nourished with bioengineered additives, such as growth factors and cell adhesion molecules, the BMCs after 24 weeks differentiated into cells with characteristics of Schwann cells – a variety of neural cell that provides the insulating myelin around the axons of peripheral nerve cells. The new cells successfully regenerated axons and extended their growth farther across nerve cell gaps toward damaged nerve stumps, with healthier vascularity.

“The differentiated cells, similar to Schwann cells, contributed significantly to the promotion of axon regeneration through the tube,” explained Yamakawa. “This success may be a further step in developing artificial nerves.”

Grafting self-donated (autologous) nerve cells to damaged nerves has been widely practiced and considered the “gold standard.” However, autologous cells for transplant are in limited supply. Allologous cells, donated by other individuals, require the host to take heavy immunosuppressant drugs.

Translating dosage compensation to trisomy 21

Authors: Jun Jiang, Yuanchun Jing, Gregory J. Cost, Jen-Chieh Chiang, Heather J. Kolpa, Allison M. Cotton, Dawn M. Carone, Benjamin R. Carone, David A. Shivak, Dmitry Y. Guschin, Jocelynn R. Pearl, Edward J. Rebar, Meg Byron, Philip D. Gregory, Carolyn J. Brown, Fyodor D. Urnov, Lisa L. Hall, & Jeanne B. Lawrence

Down’s syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down’s syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a ‘chromosome 21 Barr body’. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of ‘chromosome therapy’.

Source: Nature; (07/17/13) 

New article reviews latest advances in magnetic particle tracking in cell therapy


A new article published in Regenerative Medicine reviews the latest advances in magnetic particle tracking in cell therapy, a potentially groundbreaking strategy in disease treatment and regenerative medicine.

Cell therapy is one of the most promising avenues for regenerative medicine, however, its success is restricted by a number of limitations, such as inefficient delivery and retention of the therapeutic cells at the target organ, difficulties in monitoring the safety and efficacy of the therapy, in addition to issues obtaining and maintaining therapeutic cell phenotypes.

In a review by a group from the UCL Centre for Advanced Biomedical Imaging team (London, UK), emerging and established magnetic particle-based techniques for targeting, imaging and stimulating cells in vivo are discussed, in addition to potential benefits of their application in cell-based regenerative medicine therapies the clinic.

“The magnetic control of stem cells inside the body is a fascinating and promising concept for treatment of a vast range of diseases” commented Mark Lythgoe, director of the Centre for Advanced Biomedical Imaging at UCL. “Using microscopic nanomagnets we now have the potential to image, guide and activate therapeutic cells, combining therapy and diagnosis – theranostics – creating a novel type of dual imaging/therapy’

Commissioning Editor for Regenerative Medicine, Elena Conroy, added: “This timely review provides a much needed update on the different methods by which researchers can track cells with magnetic particles and how these can be used for cell therapy. I strongly believe that this will be of great use to cell biologists in both regenerative medicine and other research areas.”

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Developmental biology

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2, 7.4

Lucy Shapiro (born July 16, 1940, New York City) is an American developmental biologist. She is a professor of Developmental Biology at the Stanford University School of Medicine. She is the Ludwig Professor of Cancer Research and the director of the Beckman Center for Molecular and Genetic Medicine.[1] She founded a new field in developmental biology, using microorganisms to examine fundamental questions in developmental biology. Her work has furthered understanding of the basis of stem cell function and the generation of biological diversity.[2] Her ideas have revolutionized understanding of bacterial genetic networks and helped researchers to develop novel drugs to fight antibiotic resistance and emerging infectious diseases.[3] In 2013, Dr. Shapiro was presented with the 2011 National Medal of Science, which is given to individuals who have demonstrated “an outstanding breadth of knowledge in their field.”[3][4]


Shapiro, PhD
Stanford University

Virginia and D.K. Ludwig Professor
Professor, Developmental Biology
Director of the Beckman Center for Molecular and Genetic Medicine
Stanford University, Palo Alto, California, USA

The Ludwig Institute for Cancer Research Ltd is an international not-for-profit organization with a 40-year legacy of pioneering cancer discoveries. The Institute provides its scientists from around the world with the resources and the flexibility to realize the life-changing potential of their work and see their discoveries advance human health. This philosophy, combined with robust translational programs, maximizes the potential of breakthrough discoveries to be more attractive for commercial development.

The Ludwig Institute conducts its own research and clinical trials, making it a bridge from the most basic questions of life to the most pressing needs of cancer care. Since its inception, the Institute has invested more than $1.7 billion of its own resources in cancer research, and has an endowment valued at $1.2 billion. The Institute’s assets are managed by the LICR Fund.

Dr. Lucy Shapiro, DF, Ph.D serves as Virginia and D.K. Ludwig Professor of Cancer Research in the Department of Developmental Biology and Director of the Beckman Center for Molecular and Genetic Medicine at the Stanford University School of Medicine where she has been a faculty member since 1989. Dr. Shapiro founded Stanford University’s Department of Developmental Biology in 1989 and served as its Chairperson from 1989 to 1997.

Lucy Shapiro Ph.D.

Co-Founder, Co-Chair of Scientific Advisory Board, Director and Member of Nominating & Corporate Governance Committee,Anacor Pharmaceuticals, Inc.


Age Total Calculated Compensation This person is connected to 46 board members in 3 different organizations across 6 different industries.

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Lucy Shapiro named 2015 commencement speaker

Using her unique worldview as both artist and scientist, Shapiro revolutionized the field of developmental biology and set the stage for the new field of systems biology.

Lucy Shapiro

Lucy Shapiro

Stanford developmental biologist Lucy Shapiro, PhD, whose unique worldview has revolutionized the understanding of the bacterial cell as an engineering paradigm, will be the commencement speaker for the School of Medicine Class of 2015.

The diploma ceremony will be held June 13 from 11 a.m. to 1 p.m. on Alumni Green, followed by a luncheon at 1 p.m. on the Dean’s Lawn.

Shapiro, the Virginia and D. K. Ludwig Professor, has spent her career on the leading edge of developmental biology. She is the recipient of numerous awards, including the National Medal of Science in 2012 and the 2014 Pearl Meister Greengard Prize, which celebrates the achievements of outstanding women in science.

Shapiro, director of the Beckman Center for Molecular and Genetic Medicine, has been a faculty member since 1989, when she founded the medical school’s Department of Developmental Biology.

A painter who studied both biology and the fine arts as an undergraduate, Shapiro said that she sees science as part of the world of art. She began her career as a scientist focused on finding new ways of looking at and understanding living things, much as an artist does. She started by hunting for the simplest organism she could find — a bacterial cell — and then studying its molecular mechanisms. Her research into the genetic circuitry of these cells paved the way for new antibiotics. Her use of the microorganism as a model also set the stage for the emerging field of systems biology.

She has served in advisory roles in both the Clinton and George W. Bush administrations on the threat of infectious disease in developing countries. She has said that increasing levels of both antibiotic resistance and novel infectious agents are likely to be a larger threat to the world than bioterrorism. Shapiro also started a biotech company to test and develop antibiotic and antifungal medications.

Use science to make world a better place, graduates told

At the medical school’s commencement, Lucy Shapiro described how years of solitary work in the laboratory led her to influence public policy and battle the growing threat of infectious disease on the global stage.

JUN 152015


Commencement speaker Lucy Shapiro discussed how she raised alarms about the threat of emerging infectious diseases, drug-resistant pathogens “and a poor to nonexistent drug pipeline.”
Norbert von der Groeben

Developmental biologist Lucy Shapiro, PhD, told the 2015 School of Medicine graduates how, as a basic scientist who spent most of her life studying single-celled bacteria, she stepped out of her laboratory and onto the global stage to try to help the world avert a potential disaster.

“About 15 years ago, I sat up and looked around me and found that we were in the midst of a perfect storm,” said Shapiro, the Virginia and D. K. Ludwig Professor, speaking at the school’s commencement June 13 on Alumni Green. “There was a global tide of emerging infectious diseases, rampant antibiotic and antiviral resistance amongst all pathogens and a poor to nonexistent drug pipeline.

“For me the alarm bells went off, and I was convinced that I had to try and do something. Let me tell you the story of how I stepped out of my comfort zone. I launched a one-woman attack.”

She took any speaking engagement she could get to educate the public about antibiotic resistance; walked the corridors of power in Washington, D.C., lobbying politicians about the dangers of emerging infectious diseases; and used discoveries from her lab on the single-celled Caulobacter bacterium to develop new, effective disease-fighting drugs.

Bench-to-bedside for a better world

A recipient of the National Medal of Science, Shapiro exhorted the graduates to be unafraid of breaking out of their comfort zones and to use science to improve the human condition. Bridging the gap between the lab and the clinic can make the world a better place, she said.

Lloyd Minor, MD, dean of the School of Medicine, also emphasized the importance of bench-to-bedside work in his remarks to the graduates. There has never been a better time for shepherding advances in basic research into the clinic, he said.


Kristy Red-Horse, assistant professor of biology, hoods Katharina Sophia Volz, the first-ever graduate of the Interdepartmental Program in Stem Cell Biology and Regenerative Medicine.
Norbert von der Groeben

“You are beginning your careers at an unprecedented time of opportunities for biomedical science and for human health,” he said.

This year’s class of 195 graduates comprised 78 students who earned PhDs, 78 who earned medical degrees and 39 who earned master’s degrees. It included Katharina Sophia Volz, the first-ever graduate of the Interdepartmental Program in Stem Cell Biology and Regenerative Medicine — the first doctoral program in the nation focusing on stem cell science and translating it to patient care.

Volz, whose work in the lab has opened the doors to improvements in clinical care for heart patients, said Stanford Medicine is the place to be for scientists who want to make a difference in the world.

“Everybody here is reaching for the stars. We can do the best work here of anywhere,” said Volz, 28, a native of Ulm, Germany, the birthplace of Albert Einstein. She has worked in 10 different labs across the globe. Her father and mother, Johannes and Luise Volz, traveled from Germany to celebrate with her.

“I’ve never been in a more supportive environment,” said Volz, who discovered the progenitors to the muscle layer around the coronary arteries, a finding with implications for regenerative medicine and finding treatments for coronary artery disease.

Well-wishers, garlands and fussy babies

Some in the crowd of well-wishers, seated under a giant white tent, held garlands of flowers for the graduates, while toddlers ran around the lawn and babies fussed and cried. The two student speakers added humor and pathos to the occasion, with memories of their years of hard work and discovery.

“I’d like to run one last experiment,” said Francisco Jose Emilio Gimenez, a PhD graduate in biomedical information. “Who here had serious doubts they would ever finish their PhD?”


Brook Barajas, who earned a PhD in cancer biology, holds her 15-month-old son Sebastian.
Norbert von der Groeben

The dozens of hands shooting up from the stage were followed by laughter from the crowd.

Meghan Galligan, a medical degree graduate, said she was both nervous to be in front of the crowd and concerned about whether her puffy black graduation cap would stay put. “I’m wearing a French pastry hat and worried it’s going to fall off,” she said.

Her years of education to become a physician changed the day she entered clinical care training. “From the day we started clinics, we would really never be the same as those bright-eyed individuals who gathered here for orientation,” she said. “How could we be after gaining such privileged access into the human condition?”

Role as government adviser

Shapiro’s desire to improve the human condition led her out of the lab to the nation’s capital. She has since served in advisory roles in the administrations of Bill Clinton and George W. Bush on the threat of infectious disease in developing countries. Now director of the Beckman Center for Molecular and Genetic Medicine at Stanford, Shapiro has been a faculty member since 1989. She was founding chair of the Department of Developmental Biology and also started a biotech company in Palo Alto to test and develop antibiotics and antifungals.

Her lab at Stanford made breakthroughs in understanding the genetic circuitry of simple cells, setting the stage for the development of new antibiotics. Shapiro told the audience that over the 25 years that she has worked at the School of Medicine, she has seen a major shift in the connection between those who conduct research in labs and those who care for patients in clinics.

“We have finally learned to talk to each other,” Shapiro said. “I’ve watched the convergence of basic research and clinical applications without the loss of curiosity-driven research in the lab or patient-focused care in the clinic.”


Monica Eneriz-Wiemer, who earned a medical degree, hugs her mother Gloria Eneriz on June 13 before the School of Medicine’s diploma ceremony.
Norbert von der Groeben

This new connection, she said, is key to the future of global health.

“This is no ordinary time, from shattering political unrest in the Middle East and North Africa and the consequent flood of immigrant populations that serves as a petri dish for infectious pathogens, to global shifts in urban environments, to climate change, which is having substantial impact on health … all contributing to the appearance of old pathogens in new places and new pathogens for which we have no immunity.

“We here must care about an Ebola outbreak 8,000 miles away in West Africa; we here must care about a cholera outbreak in Haiti; we wait for the consequences of the earthquake in Nepal. We live in a global village.”

This is your time to shape the future, Shapiro told the graduates.

“Step out of your comfort zone and follow your intuition,” she said. “Don’t be afraid of taking chances. Ask, ‘How can I change what’s wrong?’ ”

In closing remarks, Laurie Weisberg, MD, president of the Stanford Medicine Alumni Association and clinical professor of medicine at UC San Francisco, also encouraged students to step outside of their comfort zone.

“You may be the most brilliant, creative and productive scientist, clinician, writer or entrepreneur, but you’ll never know if you don’t embrace uncertainty, take on a new challenge, and give it a try,” she said.  “To do what you love, and do it well, with all your heart — that’s what most important.

Stanford Medicine integrates research, medical education and health care at its three institutions – Stanford University School of Medicine, Stanford Health Care (formerly Stanford Hospital & Clinics), and Lucile Packard Children’s Hospital Stanford.

http://www.youtube.com/watch%3Fv%3D9xiPLvJnmhU  Feb 8, 2013

Lucy Shapiro, Stanford University – National Medal of Science 2011 for the pioneering discovery that the bacterial cell is controlled by an …


Elaine Fuchs, Ph.D.
Investigator, Howard Hughes Medical Institute
Rebecca C. Lancefield Professor
Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development

Skin harbors our largest reservoirs of stem cells. To maintain the body barrier, epidermis constantly self-renews and hair follicles undergo cyclical bouts of activity. Both stem cell compartments participate in repairing tissue damage after injury. Dr. Fuchs studies where adult stem cells come from, how they make tissues, how they repair wounds and how stem cells malfunction in cancers. Her group focuses on the mechanisms that impart skin stem cells with the ability to self-renew, develop and maintain tissues, and how these cells respond to external cues, and depart from their niche to accomplish these tasks.

Nature Reviews Genetics 13, 381 (June 2012) |   http://dx.doi.org:/10.1038/nrg3252

The 2012 March of Dimes Prize in Developmental Biology has been jointly awarded to Elaine Fuchs, of the Rockefeller University and Howard Hughes Medical Institute, and to Howard Green, of Harvard Medical School, for their pioneering research on the molecular workings of skin stem cells and inherited skin disorders. The prize recognizes researchers whose work has contributed to our understanding of the science that underlies birth defects.

Elaine Fuchs

Fiona Watt


http://dx.doi.org:/10.1242/​jcs.01408  Oct 1, 2004 J Cell Sci 117, 4877-4879.

Elaine Fuchs was born in the United States and raised just outside Chicago. In 1972 she graduated with a B.S. and highest distinction in the Chemical Sciences from the University of Illinois. Her undergraduate thesis research in physical chemistry focused on the electrodiffusion of nickel through quartz. She moved from Illinois to Princeton University to study for her PhD in Biochemistry, investigating changes in bacterial cell walls during sporulation in Bacillus megaterium. In 1977, she joined Howard Green, then at Massachusetts Institute of Technology (MIT), for her postdoctoral studies. There, she focused on elucidating the mechanisms underlying the balance between growth and differentiation in epidermal keratinocytes, a system and research area that continues to fascinate her today. In 1980, she was recruited to the University of Chicago, where she moved up through the ranks to the position of Amgen Professor of Molecular Genetics and Cell Biology and Investigator of the Howard Hughes Medical Institute. She moved to The Rockefeller University in 2002, where she is now the Rebecca C. Lancefield Professor of Mammalian Cell Biology and Development.

Elaine’s research has encompassed identifying and characterizing the keratin genes expressed in human skin, understanding the transcriptional mechanisms underlying gene expression and differentiation in the epidermis and hair follicles, and revealing roles for Wnt and BMP signaling in skin. Currently, her lab’s focus is on understanding the niche for multipotent stem cells in skin. The thread that ties her research areas together is epithelial morphogenesis, understanding how external cues transmit their signals to elicit changes in transcription, cytoskeletal architecture and adhesion to establish the epidermis and hair follicles.

In the interview that follows, Fiona Watt, Editor-in-Chief of JCS, asks Elaine about her experiences as a woman in science.

FMW:How has your research career impacted on your personal life and vice versa?

EF: My father was a geochemist who specialized in meteorites at Argonne National Laboratories. My aunt, who lived in the house next door, was a biologist at Argonne, and an ardent feminist. My sister, four years my senior, is now a neuroscientist. My mother is a housewife, who loves gardening and cooking and used to play piano and paint in oils. Growing up in such a family, and with farm fields, creeks and ponds in the near vicinity, I developed a deep interest in science that has carried me through my professional life.

If I think back to the family influences that shaped my choice of career, I remember that my Dad strongly advocated my being an elementary school teacher. My aunt, his sister, was denied admission to medical school and she encouraged me to go into medicine. My mom told me that she thought I was a good cook and therefore I should become a chemist. My older sis was my idol, although I found her intelligence intimidating. She thought I should become an anthropologist. So, in contrast to my close friend and former colleague Susan Lindquist (now director of the Whitehead Institute at MIT), I was strongly encouraged by my family to go to college and do something with my life. I chose the University of Illinois at Urbana (my Dad told me that if there was a good reason why he should spend more than $2000 a year on my education, we should sit down and discuss the matter – otherwise, I should select either University of Illinois, our State school, or University of Chicago, where he got a tuition break. I vowed NOT to go to University of Chicago, because my sis, Dad and aunt went there, and I wanted to be different).

At the University of Illinois, I was one of three women in an undergraduate physics class of 200. My perception (shaped at least in part by the general aura of the scientific community at the time) was that, if I was to be accepted as a smart student, I probably needed to perform near or at the top of my class. I subsequently began studying very long hours, forgoing sleep and even studying while eating meals in the student cafeteria and while picketing classes during Vietnam War protests. Although my near perfect performances on tough physics and chemistry exams may have turned a few heads, I don’t feel that it served the deeper purpose of education, nor did it instill in me a long-standing love for these fields.

Elaine Fuchs in her lab in 1980.

By contrast, my participation in Vietnam War protests had a deeper impact on me, and I decided to apply to the Peace Corps. Having spent my electives taking Spanish and Latin American history, I was hoping to get accepted to go to Chile, which was headed by Allende, a liberal democratic Marxist. I was instead accepted to Uganda, which was headed by Idi Amin, a ruthless tyrant. It was then that I began in earnest contemplating graduate school, choosing Princeton’s Biochemistry Department, to move from physical sciences into the realm of more medically oriented science. I always suspected that my father was somehow behind the decision by the Peace Corps to send me to Uganda, but in the end going to graduate school was probably the right choice for me.

Not having taken biology since high school, I gravitated towards the most chemically oriented labs at Princeton. When I went to visit Bruce Alberts, he informed me that he only took the best students, which I was certain did not mean me. Marc Kirschner was no longer focused on physical biochemistry, but instead had begun working with disgusting-looking frogs. I settled on a Professor who had been quite open about his views that women should not be in science. Despite the fact that I was viewed by my mentor as a major disappointment relative to a fellow male graduate student who joined the same lab, I did learn from my mentor how to do well-controlled experiments, for which I’ve been forever grateful. Twenty years later, my mentor’s views regarding my relative lack of scientific skills even seemed to soften a bit.

Although I received my PhD in biochemistry, my education had not been very typical. I graduated without yet isolating protein, RNA or DNA. However, I had been frugal with my $3000/year graduate stipend, and had managed to travel (3rd class) through India, Nepal, Guatemala, Mexico, Peru, Bolivia, Ecuador, Turkey, Greece and Egypt (I’ve still never gotten to Chile or Uganda). In retrospect, I understand why my advisor had not taken me seriously!

Somehow, I managed to be accepted into the lab of Howard Green at MIT, and during my postdoctoral years, I limited my travel to Morocco, and began in earnest doing experiments. I chose Howard’s lab, because he was one of the pioneers in mammalian stem cell biology. He had developed methods to culture human epidermal stem cells under conditions where they could be maintained and propagated. I was yearning to switch model systems from bacteria to humans, hoping that my research might be more medically applicable, and I wanted to study the biochemical mechanisms underlying the balance between growth and differentiation in normal human cells. The system seemed ideal, and led me to become a skin biologist. Mouse genetics came later in my career after I was appointed to the HHMI at the University of Chicago, and had the resources to complement the culture system.

My experience at MIT had a powerful impact on my career. Howard Green was a quintessential cell biologist, which was something completely new to me. Nearly every lab at MIT was humming with brilliant postdocs, and I rapidly got hooked on the excitement of the science around me. I began to think that perhaps a scientific career might even be a possible goal for me – at least at some small teaching college or state university. After my first year at MIT, my advisor from Princeton nominated me for an Assistant Professorship at the University of Chicago, something that I assumed was to be a trial run for an academic job later down the road. I viewed the invitation to speak as a free trip home to visit my family, and was quite amazed when I subsequently received a job offer. It was only then when I began to realize that somebody must think more highly of my accomplishments than I did. My family’s pressure to accept the position was relentless and so I began an academic career as an independent scientist, feeling at the base of a totem pole of fantastic colleagues.

FMW:What changes for women in science have you observed during the course of your career?

EF: At Chicago, I was the first woman in a department of 15 biochemistry faculty members. But Janet Rowley, who already was a member of the National Academy of Sciences and a famous cytogeneticist, was in the Department of Medicine, and she sent hand-written notes congratulating me on every small success that would come my way. This inspired me, as did meeting Susan Lindquist in the Department of Biology, who became my long-standing close friend and colleague. In 1982, Sue also introduced me to David Hansen, to whom I have been happily married for 16 years!

Chicago reorganized their biological sciences departments in 1985, and Sue, Janet, several other women and I all chose to join the same Department, Molecular Genetics and Cell Biology. All of a sudden, women faculty members were in abundance and a force to be reckoned with. This and fantastic students became an endless source of enjoyment for me, and I remained at Chicago for over 20 years.

I feel that although there is still considerable work to be done to pave the way for women in science, the situation has improved considerably during the course of my career. Women are now routinely asked and elected to serve the scientific community in important ways. In this regard, I have served on the Advisory Council for the Director of the NIH, the Council of the National Academy of Sciences (NAS) and was President of the American Society of Cell Biology. In addition, major scientific organizations have cracked the door open wider for women, and I certainly feel fortunate to have been elected by my colleagues to the NAS, the Institute of Medicine and the American Academy of Arts and Sciences. I also feel honored to have received recognition from my colleagues through a number of scientific achievement awards, including the Richard Lounsbery Award from the NAS and an honorary doctorate degree from Mt Sinai and New York University Medical School. As women continue to make their way in the scientific community at all levels and in greater numbers, we will continue to see a rise in the creativity, reflection and breadth of thinking that is so necessary to move science forward.

FMW:Do you feel that being a woman is an inherent advantage/disadvantage for a career in science? Why?

EF: I can’t say that it is or isn’t, but for me the discrimination I have faced personally has served as an inspiration and a challenge to do better, not as an impediment to my career. The one thing I do feel now is that it is important for senior women to remember that the road for women scientists is not always an easy one. There is still substantial room for the scientific community to grow in the realization that, by opening the door to women, it is going to raise the level of scientific excellence. Senior women who are recognized by their peers as being successful have a responsibility to help educate those scientists who haven’t quite accepted this important message. And we have a responsibility to maintain the highest scientific and ethical standards and to serve as the best role models we can for the younger generation of outstanding scientists – both men and women – who are rising through the ranks. Leading by good example is still the best way to diffuse the now more subtle and less vocal, but nevertheless lingering, discrimination and dogmatism against women scientists within our scientific community.

No discussion of women and careers is complete without addressing the issue of children and motherhood. In my case, I’m afraid I don’t serve as a good role model because I don’t have children. However, I’d like to emphasize that this was a decision that my husband and I consciously made together. I’m married to the Director of Philosophy and Education at Teachers’ College, Columbia University, and for the past 20 years that we’ve known one another, we’ve enjoyed traveling the world, going to operas, symphony and chamber music concerts, eating leisurely dinners, dancing, swimming, quiet reflection, education and service to the broader community. We love our nieces and nephews, but children were not a high priority for our lives together. In another world, things might be different. However, I certainly don’t view this decision as a sacrifice that I had to make for my science.

FMW:What are your remaining career ambitions?

EF: I made the decision to move to Rockefeller in 2002 because it provided an exceptional constellation of world-renowned colleagues, generating a rich and stimulating new environment for the 17 postdocs and technicians who moved with me. Our research has progressively moved to the field of morphogenesis – understanding the molecular process that begins with a single stem cell and ends with a functional tissue, either epidermis or hair follicles. Characteristic of my checkered past, the research is a blend of biochemistry, molecular biology, cell and developmental biology, and the area enables us to combine our interests in signal transduction, transcriptional regulation, cytoskeletal dynamics and cell adhesion. The caliber of my students and postdocs keeps escalating, and the science continues to keep me in the lab nights and weekends, as it did when I was a postdoctoral fellow. Each day brings new challenges, and there is certainly no doubt now that the flame of excitement and interest in scientific discovery and education burns eternally within me. There is no `last’ objective – only new horizons and challenges. The revolution in biology that I have experienced in my own career tells me not to predict what my next objective will be.

I feel strongly that we make of our lives what we put into them. To succeed in a scientific career in academia takes motivation, commitment, effort, thought, creativity, intelligence, teaching skills, technical talent, organization, leadership, oral and writing skills, compassion and a strong sense of ethics. I know I’ve left out many other essential traits. Very few scientists have all these attributes, but we can each achieve a high degree of satisfaction if not success through honing the subset of attributes that we do have. I know that for me, the more I work on becoming a better scientist, mentor and participator in our scientific community, the richer all aspects of my life become.

Elaine Fuchs: A love for science that’s more than skin deep

JCB Dec 28, 2009;  187(7): 938-939  http://dx.doi.org:/10.1083/jcb.1877pi

Elaine Fuchs has collected many awards in her 30 years researching mammalian skin development, but it’s hard to beat the two prizes she received in late 2009. Shortly before winning the prestigious L’Oreál-UNESCO award for women in science, Fuchs was awarded the National Medal of Science—the US’s highest honor for outstanding scientific contributions.

After studying bacterial sporulation as a PhD student with Charles Gilvarg at Princeton, Fuchs joined Howard Green’s laboratory at MIT, where she investigated the expression of keratins in differentiating skin cells (1, 2). Fuchs then returned to her native Illinois to begin her own laboratory at the University of Chicago, and stayed for more than 20 years before moving to The Rockefeller University in New York in 2002. Fuchs’ research has touched on many aspects of skin differentiation and function. Asked to pick her favorite work, she chooses her pioneering use of mouse genetics to identify mutant keratins as the cause of several human skin diseases (3, 4). She also mentions the generation of super furry mice by expressing a stabilized version of the transcription factor β-catenin (5) as well as the identification and characterization of a multipotent stem cell population in the hair follicle (6, 7). In a recent interview, Fuchs discussed her latest awards, and explained why the skin continues to hold her interest.


Elaine Fuchs

Is it true that you refused to take the exam for graduate school entry?   

Yes! [laughs] I was graduating near the top of my class from a very good university and I felt that the Graduate Record Examination wasn’t testing my real knowledge, but rather how I could perform in a written exam. So I decided that perhaps they’d appreciate some creative writing instead. I wrote three pages explaining the reasons why I was not going to be taking my GRE, and I sent it along with my applications.

I got accepted everywhere, but it’s quite unlikely that I would be admitted to any graduate program in the US today. I don’t think professors are as open-minded toward rebellious students as they were during the Vietnam War era.

How did you decide to go to Howard Green’s laboratory for your postdoc?

I had been working on bacterial sporulation and, in the course of that, I studied bacterial cell walls. Many antibiotics target the enzymes that synthesize cell walls, and that medical aspect was what I really liked about my science.

To maintain my interest in biomedical research, I decided to switch to the growth and differentiation of human cells, but I knew I was going to need a good culture system. Howard was a cell culture guru—he developed the use of human epidermal cells as well as the 3T3L1 line for adipocyte differentiation. Almost everyone else was using transformed mammalian cells at the time and I thought these were great systems to study—I still do.

And you’ve worked on skin ever since—what has captivated you for so long?

Skin is such a complex organ. We focus on the epithelium, but epithelial–mesenchymal interactions are very important in dictating whether keratinocyte stem cells will stratify to make an epidermis or differentiate into a sebaceous gland or hair follicle. How does that happen? How do you start with a stem cell and build a tissue? There are lots of facets to the problem, ranging from transcription to cell–cell and cell–substratum interactions. There’s this endless array of signals from the environment that, in a sense, encompasses almost every aspect of biology.

So even though we still work on skin as a model system, we continue to ask different questions. We spent 10 years working on keratins, but if I’d stuck with that, I might have burned myself out. I learned early on in my career that it’s important to choose a problem you’re interested in, even if you don’t yet know the technology you need to address it. I think people get into ruts when they become very good at something and do it over and over again. What we’re doing now is very different to what we were doing several years ago, and we continue to try novel and original approaches.

One of those original approaches was using transgenic mice to link keratins with human genetic diseases…

After cloning and sequencing the first keratins, we’d begun to hone in on the key residues that were critical for the assembly of keratin intermediate filaments, but we couldn’t predict the disease we should be looking at from the disrupted keratin networks we saw in our cultured skin cells. We thought that engineering mice harboring our dominant-negative keratin gene might offer us better clues. We set up transgenic mouse technology, but when we got our mice expressing mutant keratin, they showed no phenotype at all. I thought, “We just wasted all this time learning this technology, and we’re getting nowhere.”

Then one day a technician said, “There’s this dead mouse that’s half eaten, and it looks like it’s got a severe problem with its skin.” We took a look and it was expressing whopping amounts of our transgene. We realized that the mom was eating every single phenotypic mutant while leaving behind all the nonphenotypic ones. I gave [laboratory members] Bob Vassar and Pierre Coulombe my office for the night, and they babysat until the moms delivered. After their preliminary analysis, we sat down with a dermatology textbook and it was pretty clear: the pathology matched perfectly with epidermolysis bullosa simplex, a blistering skin disorder in humans.

But not everyone believed you at first?

No. I don’t blame people because diagnosing mice as having a particular human disease was unconventional at the time. I presented the work at a large meeting, and the chair took the microphone and said, “I don’t know what you’ve got, but you certainly don’t have EBS.” It took a few moments for me to react—it was looking pretty bad. The audience listened to the chair, who continued to declare confidently that our findings were rubbish.“There’s this endless array of signals from the environment that encompasses almost every aspect of biology.”

But at that point Mina Bissell stood up and said, “I don’t know whether she’s going to be right or wrong, but I just heard an interesting story, and I think we should give her the chance to find out.” This broke the ice for UPenn’s chair of dermatology, John Stanley, to stand up and say, “Actually, I would also diagnose the pathology as EBS.” Eight months later, we published a paper documenting the human genetic basis of EBS, so it didn’t take long to prove our hypothesis.

You were one of very few female group leaders when you began in Chicago. How was that?

A technician from another laboratory came down as I was setting up my laboratory, and said, “Are you Dr. Fuchs’ new technician?” and I had to say, “I am Dr. Fuchs!” There were cases where I’d be introduced to the seminar speaker as the prettiest member of the department—things that would make me cringe. I didn’t know what to make of these comments, and I’m not sure the men knew what to make of having me there.

I didn’t care what my salary was—it was more than I’d got as a postdoc— until after I was a tenured faculty member, when I discovered that my salary was actually lower than what they were offering to starting assistant professors. It was only after I realized I’d been underpaid all those years that I got angry. So there were definitely gender issues that could’ve distracted me, but I was so thrilled to be able to do my science that nothing else seemed to matter so much.

You’ve been a strong advocate for women in science, which was recognized by your L’Oreál-UNESCO award. Do any significant challenges remain?

Things are enormously better, particularly in the US. In general, the door is open for women all the way up to being an associate professor but it’s still difficult at the upper end of the scale—there are very few women in leadership positions. And there are still women at some universities who feel they are underpaid, have less space, and receive fewer privileges than their male colleagues. Most major universities have gotten the message, but I’m not sure all the smaller universities have followed suit.

The other prize you won recently was the National Medal of Science. How was your trip to the White House?


Fuchs receives the National Medal of Science from President Obama.


Having the President of the United States shake my hand and place a medal around my neck was a moving experience. It was also nice to have not only my husband, but also my mother (who’s close to 88 years old now), my sister, and eldest nephew present. It was particularly thrilling for me because President Obama recognizes the importance of basic research and science education to the future of our country.

Could scientists do a better job of communicating the importance of their work?

Yes—we need to educate politicians about the importance of basic research and increasing the budget for it. [Former congressman] John Porter, at a recent Howard Hughes meeting, asked us all, “When was the last time you contacted a politician and invited them to your laboratory? They need to see what scientists are doing.” If politicians don’t understand what we can learn from basic research and appreciate its importance, why should they support it?

How do you maintain your enthusiasm?

A professor’s role is a combination of research and education. I empathize with the pain students feel as they initially struggle with scientific research, yet there’s nothing more gratifying than watching a student’s first experiment work. You see them think, “Well, it’s really worth it after all. I can do it.” As long as I’m passionate about the scientific questions we tackle, I don’t think I’ll ever get tired of being a professor. It’s the best possible job in the world.

What can we expect next from the Fuchs laboratory?

New approaches, of course! We’ve identified lots of new genes that change their expression patterns as stem cells make epidermis and hair follicles. But we can’t use classical genetics to figure out what all these changes mean—a conditional knockout mouse takes a couple of years to make, and there’s a lot of redundancy in the genome. We’re developing new strategies to make functional analyses of mouse skin development a more tractable process. There are many signaling pathways that must converge to build and maintain tissues during normal development and wound repair, and a lot of pathways go awry to generate the myriad of human skin disorders, including cancers. We know a little bit here and there, yet we still have a lot of pieces to fill in. But I love the puzzle!


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