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Stem Cell Information – National Institutes of Health

By Adult Stem Cells

Introduction: What are stem cells, and why are they important? What are the unique properties of all stem cells? What are embryonic stem cells? What are adult stem cells? What are the similarities and differences between embryonic and adult stem cells? What are induced pluripotent stem cells? What are the potential uses of human stem cells and the obstacles that must be overcome before these potential uses will be realized? Where can I get more information?

Human embryonic and adult stem cells each have advantages and disadvantages regarding potential use for cell-based regenerative therapies. One major difference between adult and embryonic stem cells is their different abilities in the number and type of differentiated cell types they can become. Embryonic stem cells can become all cell types of the body because they are pluripotent. Adult stem cells are thought to be limited to differentiating into different cell types of their tissue of origin.

Embryonic stem cells can be grown relatively easily in culture. Adult stem cells are rare in mature tissues, so isolating these cells from an adult tissue is challenging, and methods to expand their numbers in cell culture have not yet been worked out. This is an important distinction, as large numbers of cells are needed for stem cell replacement therapies.

Scientists believe that tissues derived from embryonic and adult stem cells may differ in the likelihood of being rejected after transplantation. We don't yet know for certainwhether tissues derived from embryonic stem cells would cause transplant rejection, since relatively few clinical trialshave testedthe safety of transplanted cells derived from hESCS.

Adult stem cells, and tissues derived from them, are currently believed less likely to initiate rejection after transplantation. This is because a patient's own cells could be expanded in culture, coaxed into assuming a specific cell type (differentiation), and then reintroduced into the patient. The use of adult stem cells and tissues derived from the patient's own adult stem cells would mean that the cells are less likely to be rejected by the immune system. This represents a significant advantage, as immune rejection can be circumvented only by continuous administration of immunosuppressive drugs, and the drugs themselves may cause deleterious side effects.

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Stem Cell Information - National Institutes of Health

Somatic Cell Nuclear Transfer | Knoepfler Lab Stem Cell Blog

By Somatic Stem Cells

Advances in therapeuticcloning reported in the past year have been very exciting.

Somatic cellnuclear transfer (SCNT) can be used to produce very powerful human embryonic stem cells (ESC).

These new cells are called NT-ESCs for short. Neither embryos norreprogramming factors are needed to produce human NT-ESCs.Seehere,hereandherefor discussions of the pioneering papers reporting creation of NT-ESC including the first paper by the lab of Shoukhrat Mitalipov of OHSU, which I called the stem cell event of the year for 2013.

Now that human NT-ESC are a reality, the big question is how good these cells are compared to existing alternatives. For example, can they compete with induced pluripotent stem cells (IPSC) in terms of clinical impact as a basis for regenerative medicine?

Because NT-ESC are extremely difficultto make and have other issues (more on that below), the general sense in the field is that NT-ESC have to be clearly better than IPSCs in some concrete way to be a major, meaningful clinically relevant advance. Otherwise, whats the point of going to all that trouble to make them when IPSCs are relatively so easy to make?

Just a few months ago it seemed that NT-ESC might jump that high hurdle.

Mitalipovs team published aNaturepaper in July (Ma, et al) claiming that NT-ESC are demonstrably superior to IPSC. You read see my review of that paperherein whichI was pretty excited.

However, nowa new, very important paperfrom Dieter Eglis lab just came out in Cell Stem Cellreporting a very different result than that of the Ma paper.The new paper (Johannesson, et al; see graphical abstract above)conclusively shows that NT-ESC and IPSC are extremely similar cell types.So Johannesson, et al say that NT-ESCs are not better than IPSCs.Drs. Mitalipov and Ma are authors on the new paper as well that seems to contradict their own July NT-ESC paper.

We are left with a dilemma.

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Somatic Cell Nuclear Transfer | Knoepfler Lab Stem Cell Blog

Adult stem cell – ScienceDaily

By Somatic Stem Cells

Adult stem cells are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues.

Also known as somatic stem cells, they can be found in children, as well as adults.

Research into adult stem cells has been fueled by their abilities to divide or self-renew indefinitely and generate all the cell types of the organ from which they originate potentially regenerating the entire organ from a few cells.

Unlike embryonic stem cells, the use of adult stem cells in research and therapy is not controversial because the production of adult stem cells does not require the destruction of an embryo.

Adult stem cells can be isolated from a tissue sample obtained from an adult.

They have mainly been studied in humans and model organisms such as mice and rats.

The rigorous definition of a stem cell requires that it possesses two properties: Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.

Multipotency or multidifferentiative potential - the ability to generate progeny of several distinct cell types, for example both glial cells and neurons, opposed to unipotency - restriction to a single-cell type.

Some researchers do not consider this property essential and believe that unipotent self-renewing stem cells can exist.

Stem Cell Treatments Due to the ability of adult stem cells to be harvested from the patient, their therapeutic potential is the focus of much research.

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Adult stem cell - ScienceDaily

Platelet Rich Plasma Therapy, PRP Therapy, Stem Cell …

By Platelet Rich Plasma Injections

For many orthopedic injuries and conditions, Dr. Alan Lazar utilizes an alternative in-office treatment known as Regenerative Injection Therapy using Platelet Rich Plasma (PRP) injections with stem cells from fat. Currently, Dr. Lazar is one of only several physicians in the United States who is performing in-office mini liposuction usually in the abdomen to extract stem cells from fat.This fat graft is an excellent source of stem cells. Dr. Lazar combines these stem cells with blood platelets to create a PRP injection.

For more information about Platelet Regeneration Therapy in Plantation, Fort Lauderdale, Broward County, Florida, please call 954-476-9494 or request an appointment online at Alan M. Lazar, MD, FACS.

Regenerative Injection Therapy is a new non-operative treatment that enhances surgical repair and provides an option for orthopedic conditions that have traditionally required surgery or other extensive procedures. Using the patients own blood, similar to a lab test, the blood is placed in a special machine that spins the blood at high speeds, which separates the platelets from the other blood components.The platelets are then concentrated and re-injected into the injured area or arthritic joint. In turn, the platelets release substances known as growth factors that lead to improved natural tissue healing.

Platelets initiate repair and attract the assistance of stem cells. Regenerative injection therapy works by releasing the growth factors in an injured site or arthritic joint. The injected platelets release the growth factors and facilitate the process of regeneration and remodeling of the damaged area. This process is known as the healing process.

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Platelets are tiny cells that are critical to healing. They are the bodys primary source of bioactive tissue growth factors. The platelets contain thousands of growth factors, which include:

Together, these compounds control and regulate your natural healing process in response to injury and degenerative changes. By concentrating these growth factors and injecting them at the site of injury, a robust healing response is achieved.

Sometimes, a fibrin matrix is used with PRP to bridge the gap of a severely injured ligament or tendon. This provides scaffolding for new collagen to form along the damaged ligaments or tendons. This process allows even a severely damaged tissue to heal.

Platelet Rich Plasma (PRP) also attracts Mesenchymal stem cells (MSCs). MSCs are multi-potent stem cells that can differentiate into a variety of cell types during tissue repair processes. Cell types that MSCs have been shown to differentiate into include collagen secreting cells, bone forming osteoblasts and cartilage forming chondrocytes. Together these cells have the potential of rejuvenating tissues damaged by injury, degenerative changes, and osteoarthritis.

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piggyBac transposition reprograms fibroblasts to induced …

By Induced Pluripotent Stem Cells

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies.

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Platelet-Rich Plasma and BMCC (Bone Marrow Cell …

By Platelet Rich Plasma Injections

What is Platelet-Rich Plasma (PRP)? Platelet-rich plasma or PRP is an "autologous blood therapy" that uses a patient's own blood components to stimulate a healing response in damaged tissues. In response to an injury or tissue damage, your body naturally recruits platelets and white blood cells from the blood to initiate a healing response. Under normal conditions, platelets store numerous growth factors which are released in response to signals from the injured tissue. Modern technology allows us to concentrate platelets and white blood cells from your blood, and induce this growth factor release as we inject the solution directly into injured tissue, simulating this same healing response in a more powerful form. By enhancing the body's natural healing capacity, the treatment may lead to a more rapid, more efficient, and more thorough restoration of the tissue to a healthy state. What is Bone Marrow Cell Concentrate (BMCC)? It is well established that a significant population of our bodies' stem cells are contained within our bone marrow. By harvesting blood and tissue from the bone marrow space of the hip, an injectable product can be produced by concentrating platelets and cells withdrawn through a simple outpatient needle aspiration procedure, done with local anesthetic and light sedation. BMCC contains all of the growth and healing factors in PRP, along with concentrated "pluripotent" or stem-like cells which further contribute to the regenerative process. What conditions can be treated with PRP and BMCC? Knee Pain Tendon Injuries (Patellar Tendonitis, Quad Tendon) Muscle injuries Ligament sprains or tears (MCL, LCL) Bursitis Osteoarthritis Hip Pain Muscle pain or injury Pyriformis syndrome Greater Trochanteric Bursitis Tendon Injuries Sacroiliac joint pain Hamstring tendonitis or tears Osteoarthritis Shoulder and Arm Pain Rotator Cuff tendonitis, tendonopathy or partial tears Bicipital tendonitis Medial and Lateral epicondylitis (golfers & tennis elbow) Ulnar Collateral Ligament sprain or tear Lower Leg and Foot Pain Plantar Fasciitis Shin Splints Peroneal tendonitis Ankle sprains/ligament injury Achilles tendonitis or partial tears Treatment Process: Following a formal evaluation and diagnostic workup, an individualized treatment plan will be discussed with you. A full explanation of the procedure including risks and benefits will be reviewed. Once written consent is obtained, blood is drawn from your arm or from a bone marrow aspiration in the back of your hip region, and placed in a special processing unit, which separates platelets, white blood cells and serum from red blood cells. The platelets and white blood cells (including stem cells) are then concentrated and collected into a sterile syringe. Some of the blood is used to create an "activator" of the PRP/BMCC. The skin and soft tissue is anesthetized with local anesthetic, followed by injection of both the PRP/BMCC and activator into the tissue targeted for treatment. Depending on the size of the injured tissue, one or several needles are inserted to optimize placement of the product. Treatment plan: Depending on the severity and duration of your injury, one to three injections are suggested. Following the initial treatment with PRP or BMCC, a follow up visit occurs 3-4 weeks later. At this visit an evaluation of your response to the initial therapy is performed and a decision is made regarding the need for additional PRP treatments. In general, chronic injuries often require more than one injection. In both acute and chronic injuries, injections may be combined with an exercise or physical therapy program to enhance the success of the treatment. Are PRP and BMCC injections safe? Research and clinical data show that PRP and BMCC injections are extremely safe, with minimal risk for any adverse reaction or complication. Because the injectable products are produced from your own blood, there is no concern for rejection or disease transmission. There is a small risk of infection from any injection into the body, but this is rare. Of note, recent research suggests that PRP may have an anti-bacterial property which protects against possible infection (4). What to expect after your treatment: Often, following the initial injection, an "achy" soreness is felt at the site of injury. This "soreness" is a positive sign that a healing response has been set in motion. This effect can last for several days and gradually decreases as healing and tissue repair occurs. It is important that anti-inflammatory medications such as Ibuprofen, Naproxen and Aspirin be avoided following PRP treatments. These medicines may block the effects of the intended healing response facilitated by the injection itself. It is acceptable to use over the counter pain medication, such as Tylenol and in some cases a prescribed analgesic, which does not have anti-inflammatory properties, to control discomfort as needed. Pain management options will be discussed with you by the physician managing your treatment plan. You will be permitted to resume normal day to day activities and light exercise following injection. We suggest that you avoid strenuous lifting or high level exercise for at least several days after injection. How do I find out if PRP or BMCC is right for me? Questions regarding PRP or your candidacy for the treatments can be addressed to Dr. Karli at the Steadman Clinic in Vail, Colorado. Contact person for the PRP treatment program is Benjamin Dale (970) 479-2706 bdale@thesteadmanclinic.com. We would be happy to review a brief history of your problem via telephone in order to determine if a formal evaluation is warranted. Selected References 1. Anitua E, S. M., Nurden A, Nurden P, Orive G, Andia I. (2006). "New insights into and novel applications for platelet-rich fibrin therapies." Trends in Biotechnology 24(5): 227-234.

2. Mishra A, A. J., Anitua E, Andia I, Padilla S, Mujika I. (2007). "Treatment of chronic elbow tendinosis with buffered platelet-rich plasma." Am J of Sports Med 34(11): 1774-1778.

3. Moojen D, E. P., Schure R, et al. (2007). "Antimicrobial activity of platelet-leukocyte gel against Staphylococcus anreus." Journal of Orthopaedic Research DOI: 10.1002/jor.20519.

4. Sanchez M, A. E., Azofra J, Andia I, Padilla S, Mujika I. (2007). "Comparison of Surgically Repaired Achilles Tendon Tears Using Platelet-Rich Fibrin Matrices." Am J of Sports Med 10(10): 1-7.

5. Foster, T. E., Puskas, B. L., Mandelbaum, B. R., Gerhardt, M. B., & Rodeo, S. A. (2009). Platelet-rich plasma: From basic science to clinical applications. The American Journal of Sports Medicine, 2259-2272.

6. Fortier, L. A., Potter, H. G., Rickey, E. J., Schnabel, L. V., Foo, L. F., Chong, L. R., Stokol, T., & Cheetham, J. (2010). Concentrated bone marrow aspirate improves full-thickness cartilage repair compared with microfracture in the equine model. The Journal of Bone and Joint Surgery, 92(10), 1927-1937.

7. Fortier, L. A., Barker, J. U., Strauss, E. J., McCarrel, T. M., & Cole, B. J. (2011). The role of growth factors in cartilage repair. Clinical Orthopaedics and Related Research, 469(10), 2706-2715.

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Embryonic stem cell – Science Daily

By Embryonic Stem Cells

Embryonic stem cells (ESCs) are stem cells derived from the undifferentiated inner mass cells of a human embryo.

Embryonic stem cells are pluripotent, meaning they are able to grow (i.e. differentiate) into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm.

In other words, they can develop into each of the more than 200 cell types of the adult body as long as they are specified to do so.

Embryonic stem cells are distinguished by two distinctive properties: their pluripotency, and their ability to replicate indefinitely.

ES cells are pluripotent, that is, they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm.

These include each of the more than 220 cell types in the adult body.

Pluripotency distinguishes embryonic stem cells from adult stem cells found in adults; while embryonic stem cells can generate all cell types in the body, adult stem cells are multipotent and can produce only a limited number of cell types.

Additionally, under defined conditions, embryonic stem cells are capable of propagating themselves indefinitely.

This allows embryonic stem cells to be employed as useful tools for both research and regenerative medicine, because they can produce limitless numbers of themselves for continued research or clinical use.

Because of their plasticity and potentially unlimited capacity for self-renewal, ES cell therapies have been proposed for regenerative medicine and tissue replacement after injury or disease.

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Embryonic stem cell - Science Daily

Somatic cell nuclear transfer – Wikipedia, the free …

By Somatic Stem Cells

In genetics and developmental biology, somatic cell nuclear transfer (SCNT) is a laboratory strategy for creating a viable embryo from a body cell and an egg cell. The technique consists of taking an enucleated oocyte (egg cell) and implanting a donor nucleus from a somatic (body) cell. It is used in both therapeutic and reproductive cloning. Dolly the Sheep became famous for being the first successful case of the reproductive cloning of a mammal.[1] "Therapeutic cloning" refers to the potential use of SCNT in regenerative medicine; this approach has been championed as an answer to the many issues concerning embryonic stem cells (ESC) and the destruction of viable embryos for medical use, though questions remain on how homologous the two cell types truly are.

The process of somatic cell nuclear transplant involves two different cells. The first being a female gamete, known as the ovum (egg/oocyte). In human SCNT experiments, these eggs are obtained through consenting donors, many times utilizing ovarian stimulation. The second being a somatic cell, referring to the cells of the human body. Skin cells, fat cells, and liver cells are only a few examples. The nucleus of the donor egg cell is removed and discarded, leaving it 'deprogrammed.' The nucleus of the somatic cell is also removed but is kept, the enucleated somatic cell is discarded. What is left is a lone somatic nucleus and an enucleated egg cell. These are then fused by squirting the somatic nucleus into the 'empty' ovum. After being inserted into the egg, the somatic cell nucleus is reprogrammed by its host egg cell. The ovum, now containing the somatic cell's nucleus, is stimulated with a shock and will begin to divide. The egg is now viable and capable of producing an adult organism containing all the necessary genetic information from just one parent. Development will ensue normally and after many mitotic divisions, this single cell forms a blastocyst (an early stage embryo with about 100 cells) with an identical genome to the original organism (i.e. a clone).[2] Stem cells can then be obtained by the destruction of this clone embryo for use in therapeutic cloning or in the case of reproductive cloning the clone embryo is implanted into a host mother for further development and brought to term.

Somatic cell nuclear transplantation has become a focus of study in stem cell research. The aim of carrying out this procedure is to obtain pluripotent cells from a cloned embryo. These cells genetically matched the donor organism from which they came.This gives them the ability to create patient specific pluripotent cells, which could then be used in therapies or disease research.[3]

Embryonic stem cells are undifferentiated cells of an embryo. These cells are deemed to have a pluripotent potential because they have the ability to give rise to all of the tissues found in an adult organism. This ability allows stem cells to create any cell type, which could then be transplanted to replace damaged or destroyed cells. Controversy surrounds human ESC work due to the destruction of viable human embryos. Leading scientists to seek an alternative method of obtaining stem cells, SCNT is one such method.

A potential use of stem cells genetically matched to a patient would be to create cell lines that have genes linked to a patient's particular disease. By doing so, an in vitro model could be created, would be useful for studying that particular disease, potentially discovering its pathophysiology, and discovering therapies.[4] For example, if a person with Parkinson's disease donated his or her somatic cells, the stem cells resulting from SCNT would have genes that contribute to Parkinson's disease. The disease specific stem cell lines could then be studied in order to better understand the condition.[5]

Another application of SCNT stem cell research is using the patient specific stem cell lines to generate tissues or even organs for transplant into the specific patient.[6] The resulting cells would be genetically identical to the somatic cell donor, thus avoiding any complications from immune system rejection.[5][7]

Only a handful of the labs in the world are currently using SCNT techniques in human stem cell research. In the United States, scientists at the Harvard Stem Cell Institute, the University of California San Francisco, the Oregon Health & Science University,[8]Stemagen (La Jolla, CA) and possibly Advanced Cell Technology are currently researching a technique to use somatic cell nuclear transfer to produce embryonic stem cells.[9] In the United Kingdom, the Human Fertilisation and Embryology Authority has granted permission to research groups at the Roslin Institute and the Newcastle Centre for Life.[10] SCNT may also be occurring in China.[11]

In 2005, a South Korean research team led by Professor Hwang Woo-suk, published claims to have derived stem cell lines via SCNT,[12] but supported those claims with fabricated data.[13] Recent evidence has proved that he in fact created a stem cell line from a parthenote.[14][15]

Though there has been numerous successes with cloning animals, questions remain concerning the mechanisms of reprogramming in the ovum. Despite many attempts, success in creating human nuclear transfer embryonic stem cells has been limited. There lies a problem in the human cell's ability to form a blastocyst; the cells fail to progress past the eight cell stage of development. This is thought to be a result from the somatic cell nucleus being unable to turn on embryonic genes crucial for proper development. These earlier experiments used procedures developed in non-primate animals with little success. A research group from the Oregon Health & Science University demonstrated SCNT procedures developed for primates successfully reprogrammed skin cells into stem cells. The key to their success was utilizing oocytes in metaphase II (MII) of the cell cycle. Egg cells in MII contain special factors in the cytoplasm that have a special ability in reprogramming implanted somatic cell nuclei into cells with pluripotent states. When the ovum's nucleus is removed, the cell loses its genetic information. This has been blamed for why enucleated eggs are hampered in their reprogramming ability. It is theorized the critical embryonic genes are physically linked to oocyte chromosomes, enucleation negatively affects these factors. Another possibility is removing the egg nucleus or inserting the somatic nucleus causes damage to the cytoplast, affecting reprogramming ability. Taking this into account the research group applied their new technique in an attempt to produce human SCNT stem cells. In May 2013, the Oregon group reported the successful derivation of human embryonic stem cell lines derived through SCNT, using fetal and infant donor cells. Using MII oocytes from volunteers and their improved SCNT procedure, human clone embryos were successfully produced. These embryos were of poor quality, lacking a substantial inner cell mass and poorly constructed trophectoderm. The imperfect embryos prevented the acquisition of human ESC. The addition of caffeine during the removal of the ovum's nucleus and injection of the somatic nucleus improved blastocyst formation and ESC isolation. The ESC obtain were found to be capable of producing teratomas, expressed pluripotent transcription factors, and expressed a normal 46XX karyotype, indicating these SCNT were in fact ESC-like.[8] This was the first instance of successfully using SCNT to reprogram human somatic cells. This study used fetal and infantile somatic cells to produce their ESC.

In April 2014, an international research team expanded on this break through. There remained the question of whether the same success could be accomplished using adult somatic cells. Epigenetic and age related changes were thought to possibly hinder an adult somatic cells ability to be reprogrammed. Implementing the procedure pioneered by the Oregon research group they indeed were able to grow stem cells generated by SCNT using adult cells from two donors, aged 35 and 75.Indicating age does not impede a cells ability to be reprogrammed[16][17]

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Somatic cell nuclear transfer - Wikipedia, the free ...

Glossary [Stem Cell Information] – Embryonic stem cell

By Somatic Stem Cells

Adult stem cellsee somatic stem cell.

Astrocytea type of supporting (glial) cell found in the nervous system.

BlastocoelThe fluid-filled cavity inside the blastocyst, an early, preimplantation stage of the developing embryo.

BlastocystA preimplantation embryo of about 150 cells produced by cell division following fertilization. The blastocyst is a sphere made up of an outer layer of cells (the trophoblast), a fluid-filled cavity (the blastocoel), and a cluster of cells on the interior (the inner cell mass).

Bone marrow stromal cellsA population of cells found in bone marrow that are different from blood cells, a subset of which are multipotent stem cells, able to give rise to bone, cartilage, marrow fat cells, and able to support formation of blood cells.

Bone marrow stromal cellsA population of cells found in bone marrow that are different from blood cells, a subset of which are multipotent stem cells, able to give rise to bone, cartilage, marrow fat cells, and able to support formation of blood cells.

Bone marrow stromal stem cells (skeletal stem cells)A multipotent subset of bone marrow stromal cells able to form bone, cartilage, stromal cells that support blood formation, fat, and fibrous tissue.

Cell-based therapiesTreatment in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cells or tissues.

Cell cultureGrowth of cells in vitro in an artificial medium for research or medical treatment.

Cell divisionMethod by which a single cell divides to create two cells. There are two main types of cell division depending on what happens to the chromosomes: mitosis and meiosis.

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Glossary [Stem Cell Information] - Embryonic stem cell

Human oocytes reprogram adult somatic nuclei of a type 1 …

By Somatic Stem Cells

The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

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Human oocytes reprogram adult somatic nuclei of a type 1 ...

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