Direct Reprogramming Cells with Recombinant Proteins

In my opinion, for what it’s worth, we will probably see direct reprogramming take a prominent place in regenerative medicine in the future. It will not be in the near future, but as direct reprogramming becomes better understood and more feasible, it will probably become a central part of the discussion of regenerative medical strategies.

Direct reprogramming, which is also known as lineage conversion, uses cell type-specific transcription factors to convert a mature, adult cell into a different type of mature, adult cell. The cell does not pass through a pluripotent intermediate, and becomes a wholly different type of cell.

Of course, forcing the expression of lineage-specific transcription factors in cells requires that they be treated with recombinant viruses or other such tools. These genetic manipulations present problems for regenerative medicine, since such viruses can cause mutations or cause the introduced genes to be constantly activated, both of which can cause cells to die to grow uncontrollably. Genetically engineering cells needs to be done in a “kinder and gentler” way (to quote George HW Bush).

To that end Dennis Clegg and his colleagues from the Center for Stem Cell Biology and Engineering at UC Santa Barbara have used specially designed proteins to directly cultured retinal pigmented epithelial cells to neurons.

Newly discovered C-end rule (CendR) cell- and tissue-penetrating peptides have a arginine-rich sequence at the end of proteins that allows them to bind particular cell receptors and be internalized into the cell. These CendR peptides bind to the NRP-1 protein and are internalized. Several laboratories have used CendR peptides to increase the efficacy of anti-cancer drugs in experimental cases (see Alberici L, et al (2013) Cancer Res 73:804–812; Sugahara KN, et al. (2010) Science 328:1031–1035; Sugahara KN, et al. (2009) Cancer Cell 16:510–520; and Roth L,, et al. (2012) Oncogene 31:3754–3763).

By tacking a CendR peptide to the end of the Sox2 protein, Clegg and others were able to convert retinal pigmented epithelial (RPEs) cells to neurons. The Sox2 protein is highly expressed in neural progenitor cells. Other studies have shown that Sox2 can reprogram mouse and human fibroblasts to neural stem cells (Ring KL, et al. (2012) Cell Stem Cell 11:100–109). Thus, Sox2 should do the trick.

Making cultured RPE cells from embryonic stem cells is relatively easy to do. Therefore, Clegg and his coworkers made cultured RPEs and then treated them with viruses that expressed Sox2. The cultured RPEs showed conversion to neurons and the expression of neuron-specific genes.

Since they had established that Sox2 could convert RPEs to neurons, they tried recombinant Sox2 protein with the CendR peptide RPARPAR at the end of the protein. After 60 days in culture, the cells expressed a host of neuron-specific genes, and were capable of taking up a dye that only active neurons can take up (FM1-43).

Reprogramming human fetal RPE (hfRPE) cells to neurons using recombinant SOX2 proteins. (A): Efficiency of hfRPE cells to be reprogrammed to neuron-like cells after recombinant proteins was added to the media every 24 hours for 30 days. (B): Efficiency of hfRPE cells to be reprogrammed by adding SOX2-RPARPAR recombinant protein every 48 hours for different time courses. (C): Representative images of hfRPE (fRPE1914) cells during reprogramming to neuron-like cells after 30, 40, and 50 days in culture with SOX2-RPARPAR protein. Scale bars = 100 μm. (D): Representative images of hfRPE (fRPE1914) cells reprogrammed to neuron-like cells expressing neuronal markers, but not an RPE marker (PAX6), using SOX2-RPARPAR protein. Scale bars = 50 μm. Abbreviations: D, days; RPE, retinal pigmented epithelial cells.
Reprogramming human fetal RPE (hfRPE) cells to neurons using recombinant SOX2 proteins. (A): Efficiency of hfRPE cells to be reprogrammed to neuron-like cells after recombinant proteins was added to the media every 24 hours for 30 days. (B): Efficiency of hfRPE cells to be reprogrammed by adding SOX2-RPARPAR recombinant protein every 48 hours for different time courses. (C): Representative images of hfRPE (fRPE1914) cells during reprogramming to neuron-like cells after 30, 40, and 50 days in culture with SOX2-RPARPAR protein. Scale bars = 100 μm. (D): Representative images of hfRPE (fRPE1914) cells reprogrammed to neuron-like cells expressing neuronal markers, but not an RPE marker (PAX6), using SOX2-RPARPAR protein. Scale bars = 50 μm. Abbreviations: D, days; RPE, retinal pigmented epithelial cells.

The efficiency for this experiment was lousy (0.3%) as opposed to the efficiency for the use of recombinant viruses (11%). Nevertheless, this experiment shows that it is possible to directly reprogram cells without using recombinant viruses.

Putting Peps in Your Heps

The liver is a special organ that performs a whole host of essential functions. The liver stores iron, vitamins and minerals; it detoxifies alcohol, drugs, and other chemicals that accumulate in our bloodstreams, and it produces bile (used to dissolve fats so that they can be degraded), and blood-based proteins like clotting factors and albumin. The liver also stores sugar in the form of glycogen. All of these tasks are undertaken by a single cell type, the hepatocyte (otherwise known as a liver cell).


When your liver fails, you get really sick. This was greatly illustrated to me by one of my colleagues where I teach whose wife suffered extensive liver damage as a result of her battle with lupus (short for systemic lupus erythematosus, an autoimmune disease). Now that this dear lady has had a liver transplant, she is a new person. What a difference a healthy liver makes.

What can regenerative medicine do for patients with failing livers? Human pluripotent stem cells, either embryonic stem cells or induced pluripotent stem cells, can be directed to differentiate into liver cells in culture, but the liver cells made by these cells are very immature. They express proteins commonly found in fetal liver cells (for example, alpha-fetoprotein) and they also lack key enzymes associated with adult cells (such as cytochrome P450s). Rashid and others in the Journal of Clinical Investigation (2010; 120: 3127-3136) showed this. The development of three-dimensional culture systems have increased the maturity of such cells, but there is still a long way to go (see T Takebe and others, Nature 2013; 499:481-484 and J Shan and others, Nature Chemical Biology 2013; 9: 514-520).

Two papers from the journal Cell Stem Cell might show a way forward to making mature liver cells for regenerative liver treatments without destroying embryos or even using and pluripotent stem cell lines. These papers utilize the procedure known as “direct reprogramming,” otherwise known as “direct lineage conversion.” Direct reprogramming requires the forced overexpression of particular genes that causes the cells to switch their cell types.

In the first of these papers, Pengyu Huang and his colleagues from the Chinese Academy of Sciences in Shanghai, China overexpressed a three-gene combination in mouse embryonic fibroblasts that converted the cells into hepatocytes at an efficiency of 20% after 14 days in culture. This gene combination, known as 3TF (HNF4/HNF1A/FOXA3), converted the mouse embryonic skin cells into mature liver cells that made blood proteins and drug-processing enzymes. The only problem was that these mature cells could not grow in culture because they were mature. Therefore, Huang and others infected these cells with a virus called SV40, which drove the cells to divide. Now these cells could be grow in culture and expanded for further experiments.

When transplanted into the livers of mice with failing livers, the induced liver cells made by Huang and others restored proper liver function and allowed the mice to survive.

A second paper by Yuanyuan Du and others from the Peking-Tsinghua Center for Life Sciences at Peking University in Beijing, China, used a large gene combination to make mature liver cells from human skin fibroblasts. This gene combination included eight genes (HNF1A/HNF4A/HNF6/ATF5/PROX1/CEBPA/p53 ShRNA/C-MYC) that converted the human skin cells into liver cells after 30 days in culture at an efficiency of nearly 80%. Again, these cells metabolized drugs as they should, made blood proteins, took up cholesterol, and stored glycogen. Du and others compared the gene expression profile of these human induced hepatocytes or “hiHeps” to the gene expression profile of liver cells taken from liver biopsies. While there were differences in gene expression, there was also significant overlap and a large overall similarity. In fact the authors state, “these results indicate that hiHeps show a similar expression profile to primary human hepatocytes.”

Next, Du and others used three different mouse models of liver failure in all three cases, the hiHeps were capable of colonizing the damaged liver of the mouse and regenerating it. Mind you, the hiHeps did not do as good a job as human primary hepatocytes, but they still worked pretty well. This shows that this direct reprogramming protocol, as good as it is, can still be optimized and improved.

These studies show that the production of highly functional human hepatocyte-like cells using direct reprogramming is feasible and represents an exciting step towards the production of a supply source of cells for drug development, and therapies for liver disease.

Directly Reprogramming Skin Cells into White Blood Cells

Scientists from the Salk Institute have, for the first time, directly converted human skin cells into transplantable white blood cells, which are the soldiers of the immune system that fight infections and invaders. This work could prompt the creation of new therapies that introduce new white blood cells into the body that can attack diseased or cancerous cells or augment immune responses for other conditions.

This work, which shows that only a small amount of genetic manipulation could prompt this direct conversion, was published in the journal Stem Cells.

“The process is quick and safe in mice,” says senior author Juan Carlos Izpisua Belmonte, who holds the Salk’s Roger Guillemin Chair. “It circumvents long-standing obstacles that have plagued the reprogramming of human cells for therapeutic and regenerative purposes.”

The problems that Izpisua Belmonte mentions, includes the long time (at least two months) numbingly tedious cell culture work it takes to produce, characterize and differentiate induced pluripotent stem (iPS) cells. Blood cells derived from iPSCs also have other obstacles: they engraft into organs or bone marrow poorly and can cause tumors.

The new method designed by Izpisua Belmonte and his team, however, only takes two weeks, does not produce tumors, and engrafts well.

“We tell skin cells to forget what they are and become what we tell them to be—in this case, white blood cells,” says one of the first authors and Salk researcher Ignacio Sancho-Martinez. “Only two biological molecules are needed to induce such cellular memory loss and to direct a new cell fate.”

This faster reprogramming technique developed by Belmonte’s team utilized a form of reprogramming that does not go through a pluripotency stage. Such techniques are called indirect lineage conversion or direct reprogramming. Belmonte’s group has demonstrated that such approaches can reprogram cells to form the cells that line blood vessels. Thus instead of de-differentiating cells into an embryonic stem cell-type stage, these cells are rewound just enough to instruct them to form the more than 200 cell types that constitute the human body.

Direct reprogramming used in this study uses a molecule called SOX2 to move the cells into a more plastic state. Then, the cells are transfected with a genetic factor called miRNA125b that drives the cells to become white blood cells. Belmonte and his group are presently conducting toxicology studies and cell transplantation proof-of-concept studies in advance of potential preclinical and clinical studies.

“It is fair to say that the promise of stem cell transplantation is now closer to realization,” Sancho-Martinez says.

Study co-authors include investigators from the Center of Regenerative Medicine in Barcelona, Spain, and the Centro de Investigacion Biomedica en Red de Enfermedades Raras in Madrid, Spain.

Skin Cells Converted into Blood Cells By Direct Reprogramming

Making tissue-specific progenitor cells that possess the ability to survive, but have not passed through the pluripotency state is a highly desirable goal of regenerative medicine. The technique known as “direct reprogramming” uses various genetic tricks to transdifferentiate mature, adult cells into different cell types that can be used for regenerative treatments.

Juan Carlos Izpisua Belmonte and his colleagues from the Salk Institute for Biological Studies in La Jolla, California and his collaborators from Spain have used direct reprogramming to convert human skin cells into a type of white blood cells.

These experiments began with harvesting skin fibroblasts from human volunteers that were then forced to overexpress a gene called “Sox2.” The Sox2 gene is heavily expressed in mice whose bone marrow stem cells are being reconstituted with an infusion of new stem cells. Thus this gene might play a central role is the differentiation of bone marrow stem cells.

Sox2 overexpression in human skin fibroblasts cause the cells express a cell surface protein called CD34. Now this might seem so boring and unimportant, but it is actually really important because CD34 is expressed of the surfaces of hematopoietic stem cells. Hematopoietic stem cells make all the different types of white and red blood cells in our bodies. Therefore, the expression of these protein is not small potatoes.

In addition to the expression of CD34, other genes found in hematopoietic stem cells were also induced, but not strongly. Thus overexpression of SOX2 seems to induce an incipient hematopoietic stem cell‐like status on these fibroblasts. However, could these cells be pushed further?

Gene profiling of hematopoietic stem cells from Umbilical Cord Blood identified a small regulatory RNA known as miR-125b as a factor that pushes SOX2-generated CD34+ cells towards an immature hematopoietic stem cell-like progenitor cell that can be grafted into a laboratory animal.

When SOX2 and miR-125b were overexpressed in combination, the cells transdifferentiated into monocytic lineage progenitor cells.

What are monocytes? They are a type of white blood cells and are, in fact, the largest of all white blood cells. Monocytes compose 2% to 10% of all white blood cells in the human body. They play multiple roles in immune function, including phagocytosis (gobbling up bacteria and other stuff), antigen presentation (identifying and altering other cells to the presence of foreign substances), and cytokine production (small proteins that regulate the immune response).

Monocytes express a molecule on their cell surfaces called CD14, and when human fibroblasts overexpressed Sox2 and miR-125b, they became CD14-expressing cells that looked and acted like monocytes. These cells were able to gobble up bacteria and other foreign material, and when transplanted into a laboratory animal, these directly reprogrammed cells generated cells that established the monocytic/macrophage lineage.

Cancer patients, and other patients with bone marrow diseases can have trouble making sufficient white blood cells. A technique like this can generate transplantable monocytes (at least in laboratory animals) without many of the drawbacks associated with reprogramming human cells into hematopoietic stem cells that possess true clinical potential. Also because this technique skips the pluipotency stage, it is potentially safer.

Digestive Cells Converted into Insulin-Secreting Cells

By switching off a single gene, Columbia Medical Center scientists have converted cells from the digestive tract into insulin-secreting cells. This suggests that drug treatments might be able to convert gut cells into insulin-secreting cells.

Senior author Domenico Accili said this of this work: “People have been talking about turning one cell into another for a long time, but until now we hadn’t gotten to the point of creating a fully functional insulin-producing cell by the manipulation of a single target.”

Accili’s work suggests that lost pancreatic beta cells might be replaced by retraining existing cells rather than transplanting new insulin-secreting cells. For nearly two decades, scientists have been trying to differentiate a wide variety of stem cells into pancreatic beta cells to treat type 1 diabetes. In type 1 diabetes, the patient’s insulin-producing beta cells are destroyed, usually by the patient’s own immune system. The patient becomes dependent on insulin shots in order to survive.

Without insulin, cells have no signal to take up sugar and metabolize it. Also muscles and the liver do not take up amino acids and make protein, and the body tends to waste away, ravaged by high blood sugar levels that progressively and relentlessly damage it without the means to repair this damage.

Insulin-producing beta cells can be made in the lab from several different types of stem cells, but the resulting beta cells often do not possess all the properties of naturally occurring beta cells.

This led Accili and others to attempt to transform existing cells into insulin-secreting beta cells. In previous work, Accili and others demonstrated that mouse intestinal cells could be converted into insulin-secreting cells (see Talchai C, et al., Nat Genet. 2012 44(4):406-12), This recent paper demonstrates that a similar technique also works in human intestinal cells.

The gene of interest, FOXO1, is indeed present in human gut endocrine progenitor and serotonin-producing cells. In order to determine in FOXO1 inhibition could induce the formation of insulin-secreting cells, Accili and others used human induced pluripotent stem cells (iPSCs) and small “gut organoids,” which are small balls of gut tissue that grow in culture.

Inhibition of FOXO1 by either introducing a mutant version of the gene that encoded a protein that soaked up all the wild-type protein or by using viruses that forced the expression of a small RNA that prevented the expression of the FOXO1 gene caused loss of FOXO1 activity. FOXO1 inhibition promoted the generation of insulin-positive cells within the gut organoids that express all the genes and proteins normally found in mature pancreatic β-cells. These transdifferentiated cells also released “C-peptide,” which is a byproduct of insulin production, in response to drugs that drive insulin secretion (insulin secretagogues). Furthermore, these cultured insulin-secreting cells and survive when transplanted into mice where they continue to secrete insulin in response to increased blood sugar concentrations.

The findings of Accili and his colleagues provide some evidence that gut-targeted FOXO1 inhibition or transplantation of cultured gut organoids made from iPSCs could serve as a source of insulin-producing cells to treat human diabetes.

This is a remarkable piece of research, but there is one thing that troubles me about it. If the patient’s immune system has been sensitized to beta cells, making new beta cells will simply give the immune system something else to attack. It seems to me that retraining to immune system needs to be done first before replacement of the beta cells can ever hope to succeed.

Mouse Blood Cells Reprogrammed into Blood Cell Stem Cells

Boston Children’s Hospital researchers have directly reprogrammed mature blood cells from mice into blood-forming hematopoietic stem cells by using a cocktail of eight different transcription factors.

These reprogrammed cells have been called induced hematopoietic stem stem cells or iHSCs. These cells have all the hallmarks of mature mouse HSCs and they have the capacity to self-renew and differentiate into all the blood cells that circulate throughout the body.

These findings are highly significant from a clinical perspective because they indicate that it might be entirely possible to directly reprogram a patient’s existing, mature blood cells into a hematopoietic stem cell for transplantation purposes. Such a procedure, known as hematopoietic stem cells transplantation or HSCT, is a common treatment for patients whose bone marrow has suffered irreparable damage due to environmental causes (heavy metals, chloramphenicol, etc) or disease (cancer). The problem with HSCT is finding a proper match for the patient and then procuring clinically significant quantities of high-quality bone marrow for HSCT.

Derrick J. Rossi, an investigator in the Program in Cellular and Molecular Medicine at Boston Children’s Hospital and Assistant Professor in the Department of Stem Cell & Regenerative Biology, explained: “HSCs comprise only about one in every 20,000 cells in the bone marrow. If we could generate autologous (a patient’s own) HSCs from other cells, it could be transformative for transplant medicine and for our ability to model diseases of blood development.”

Rossi and his collaborators have screened genes that are expressed in 40 different types of blood progenitor cells in mice. This screen identified 36 different genes that control the expression of the other genes. These 36 genes encode so-called “transcription factors,” which are proteins that bind to DNA and turn gene express on or shut it off.

Blood cell production tends to go from the stem cells to progeny cells called progenitor cells that can still divide to some limited extent, and to effector cells that are completely mature and, in most cases, do not divide (the exception is lymphocytes, which expand when activated by specific foreign substances called antigens).

Further work by Rossi and others identified six transcription factors (Hlf, Runx1t1, Pbx1, Lmo2, Zfp37, and Prdm5) of these 36 genes, plus two others that were not part of their original screen (N-Myc and Meis1) that could robustly reprogram myeloid progenitor cells or pro/pre B lymphocytes into iHSCs.


To put these genes into these blood cells, Rossi and others uses souped-up viruses that introduced all either genes under the control of sequences that only allowed expression of these eight genes in the presence of the antibiotic doxycycline. Once these transfected cells were transplanted into mice, the recipient mice were treated with doxycycline, and the implanted cells formed HSCs.

When this experiment utilized mice that were unable to make their own blood cells, because their bone marrow had been wiped out, the implanted iHSCs reconstituted the bone marrow and blood cells of the recipient mice.

To further show that this reconstituted bone marrow was normal, high-quality bone marrow, Rossi and others used these recipient mice as bone marrow donors for sibling mice whose bone marrow had been wiped out. This experiment showed that the mice that had received the iHSCs had bone marrow that completely reconstituted the bone marrow of their siblings. This established that the iHSCs could completely reestablish the bone marrow of another mouse.

Thus Rossi and others had established that iHSCs could in fact created HSCs from progenitor cells, but could they do the same thing with mature blood cells that were not progenitor cells? Make that another yes. When Rossi and others transfected their eight-gene cocktail into mature myeloid cells, these mature cells also made high-quality iHSCs.

Rossi noted that no one has been able to culture HSCs in the laboratory for long periods of time. A few laboratories have managed expand HSCs in culture, but only on a limited basis for short periods of time (see Aggarwal R1, Lu J, Pompili VJ, Das H. Curr Mol Med. 2012 Jan;12(1):34-49).  In these experiments, Rossi used his laboratory mice as living culture systems to expand his HSCs, which overcomes the problems associated with growing these fussy stem cells in culture.

Gene expression studies of his iHSCs established that, from a gene expression perspective, the iHSCs were remarkably similar to HSCs isolated from adult mice.

This is certainly an exciting advance in regenerative medicine, but it is far from being translated into the clinic.  Can human blood progenitor cells also be directly reprogrammed using the same cocktail?  Can mature myeloid cells be successfully reprogrammed?  Will some non-blood cell be a better starting cell for iHSC production in humans?  As you can see there are many questions that have to be satisfactorily answered before this procedure can come to the clinic.

Nevertheless, Rossi and his team has succeeded where others have failed and the results are remarkable.  HSCs can be generated and transplanted with the use of only a few genes.  This is certainly the start of what will hopefully be a fruitful regenerative clinical strategy.

On a personal note, my mother passed about almost a decade ago after a long battle with myelodysplastic syndrome, which is a pre-leukemic condition in which the bone marrow fails to make mature red blood cells.  Instead the bone marrow fills up with immature red blood cells and the patient has to survive on blood transfusions.

The reason for this condition almost certainly stems from defective HSCs that do not make normal progeny.  Therefore the possibility of using a patient’s own cells to make new HSCs that can repopulate the bone marrow is a joyful discovery for me to read about, even though it is some ways from the clinic at this point.

Directly Reprogramming Gut Cells into Beta Cells to Treat Diabetes

Type 1 diabetes mellitus results from destruction of insulin-producing beta cells in the pancreas. Diabetics have to give themselves routine shots of insulin. The hope that stem cells offer is the production of cells that can replace the lost beta cells. “We are looking for ways to make new beta cells for these patients to one day replace daily insulin injections,” says Ben Stanger, MD, PhD, assistant professor of Medicine in the Division of Gastroenterology, Perelman School of Medicine at the University of Pennsylvania.

Some diabetics have had beta cells from cadavers transplanted into their bodies to replace the missing beta cells. Such a procedure shows that replacement therapy is, in principle possible. Therefore, transplanting islet cells to restore normal blood sugar levels in type 1 diabetics could treat and even cure disease. Unfortunately, transplantable islet cells are in short supply, and stem cell-based approaches have a long way to go before they reach the clinic. However, Stanger and his colleagues have tried a different strategy for treating type 1 diabetes. “It’s a powerful idea that if you have the right combination of transcription factors you can make any cell into any other cell. It’s cellular alchemy,” comments Stanger.

New research from Stanger and a postdoctoral fellow in his laboratory, Yi-Ju Chen that was published in Cell Reports, describes the production of new insulin-making cells in the gut of laboratory animals by introducing three new transcription factors. This experiment raises the prospect of using directly reprogrammed adult cells as a source for new beta cells.

In 2008, Stanger and others in Doug Melton’s laboratory used three beta-cell reprogramming factors (Pdx1, MafA, and Ngn3, collectively called PMN) to convert pancreatic acinar cells (the cells in the pancreas that secrete enzymes rather than hormones) into cells that had many of the features of pancreatic beta cells.

Following this report, the Stanger and his team set out to determine if other cells types could be directly reprogrammed into beta cells. “We expressed PMN in a wide spectrum of tissues in one-to-two-month-old mice,” says Stanger. “Three days later the mice died of hypoglycemia.” It was clear that Stanger and his crew were on to something. Further work showed that some of the mouse cells were making way too much extra insulin and that killed the mice.

When the dead mice were autopsied, “we saw transient expression of the three factors in crypt cells of the intestine near the pancreas,” explained Stanger.

They dubbed these beta-like, transformed cells “neoislet” cells. These neoislet cells express insulin and show outward structural features akin to beta cells. These neoislets also respond to glucose and release insulin when exposed to glucose. The cells were also able to improve hyperglycemia in diabetic mice.

Stanger and his co-workers also figured out how to turn on the expression of PMN in only the intestinal crypt cells to prevent the deadly whole-body hypoglycemia side effect that first killed the mice.

In culture, the expression of PMN in human intestinal ‘‘organoids,’ which are miniature intestinal units grown in culture, also converted intestinal epithelial cells into beta-like cells.

“Our results demonstrate that the intestine could be an accessible and abundant source of functional insulin-producing cells,” says Stanger. “Our ultimate goal is to obtain epithelial cells from diabetes patients who have had endoscopies, expand these cells, add PMN to them to make beta-like cells, and then give them back to the patient as an alternate therapy. There is a long way to go for this to be possible, including improving the functional properties of the cells, so that they more closely resemble beta cells, and figuring out alternate ways of converting intestinal cells to beta-like cells without gene therapy.”

This is hopefully a grand start to what might be a cure for type 1 diabetes.