Fat-derived cells Enhance the Bone-Forming Capacity of Hypertrophic Cartilage Matrices


Treating particular bone defects or injuries present a substantial challenges for clinicians. The method of choice usually involves the use of an “autologous” bone graft (“autologous” simply means that the graft comes from the patient’s own bone). However, autologous bone grafts have plenty of limitations. For example, if a patient has a large enough bone defect, there is no way the orthopedist and take bone from a donor site without causing a good deal of risk to the donor site. Even with small bone grafts, so-called “donor site morbidity” remains a risk. Having said that, plenty of patients have had autologous bone grafts that have worked well, but larger bone injuries or defects are not treatable with autologous bone grafts.

The answer: bone substitute materials. Bone substitute materials include tricalcium phosphate, hydroxyapatite, cement, ceramics, bioglass, hydrogels, polylactides, PMMA or poly(methy methacrylate) and other acrylates,, and a cadre of commercially available granules, blocks, pastes, cements, and membranes. Some of these materials are experimental, but others do work, even if do not work every time. The main problem with bone substitute materials is that, well, they are not bone, and, therefore lack the intrinsic ability to induce the growth of new bone (so-called osteoinductive potential) and their ability to integrate into new bone is also a problem at times.

We must admit that a good deal of progress has been made in this area and it’s a good thing too. Many of our fabulous men and women-at-arms have returned home with severe injuries from explosives and wounds from large-caliber weapons that have shattered their bones. These courageous men and women have been the recipient of these technologies. However, the clinician is sometimes left asking herself, “can we do better?”

A new paper from the laboratories of Ivan Martin and Claude Jaquiery from the University Hospital of Basel suggests that we can. This paper appeared in Stem Cells Translational Medicine and describes the use of a hypertrophic cartilage matrix that was seeded with cells derived from the stromal vascular faction of fat to not only make bone in the laboratory, but to also heal skull defects in laboratory animals. While this work benefitted laboratory animals, it was performed with human cells and materials, which suggests that this technique, if it can be efficiently and cheaply scaled up, might be usable in human patients.
The two lead authors of this paper, Atanas Todorov and Matthias Kreutz and their colleagues made hypertrophic cartilage matrices from human bone marrow mesenchymal stem cells (from human donors) that were induced to make cartilage. Fortunately, protocols have been very well worked out and making cartilage plugs with chondrocytes that are enlarged (hypertrophic) is something that has been successfully done in many laboratories. After growing the mesenchymal stem cells in culture, half a million cells were induced to form cartilage with dexamethasone, ascorbic acid 2-phosphate, and the growth factor TGF-beta1. After three weeks, the cartilage plugs were subjected to hypertrophic medium, which causes the cartilage cells to enlarge.

Chondrocyte enlargement is a prolegomena to the formation of bone and during development, many of our long bones (femur, humerus, fibula, radius, etc.), initially form as cartilage exemplars that are replaced by bone as the chondrocytes enlarge. Ossification begins when a hollow cylinder forms in the center of the bone (known as the periosteal collar). The underlying chondrocytes degenerate and die, thus releasing the matrix upon which calcium phosphate crystals accrete. The primary ossification center commences with the calcification of the central shaft of the bone and erosion of the matrix by the invasion of a blood vessel. The blood vessels bring osteoprogenitor cells that differentiate into osteoblasts and begin to deposit the bone matrix.

Next, Todorov and his crew isolated the stromal vascular fraction from fat that was donated by 12 volunteers who had fat taken from them by means of liposuction. The fat is then minced, digested with enzymes, centrifuged, filtered and then counted. This remaining fraction is called the stromal vascular fraction or SVF, and it consists of a pastiche of blood vessel-forming cells, mesenchymal stem cells, and bone-forming cells (and probably a few other cells types too). These SVF cells were seeded onto the hypertrophic cartilage plugs and used for the experiments in this paper.

First, the SVF-seeded plugs were used to grow bone in laboratory rodents. The cartilage plugs were implanted into the backs for nude mice. Different cartilage plugs were used that had been seeded with gradually increasing number of SVF cells. The implanted plugs definitely made ectopic bone, but the amount of bone they made was directly proportional to the number of SVF cells with which they had been seeded. Staining experimental also showed that some of the newly-grown bone came from the implanted SVF cells.

Ectopic bone formation. Grafts based on devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction cells from adipose tissue and implanted subcutaneously in nude mice. (A): Representative hematoxylin and eosin, Masson-Tri-Chrome, and Safranin-O (Saf-O) staining and in situ hybridization for human ALU sequences (dark blue = positive) after 12 weeks in vivo. Saf-O stainings are blue-green because of lack of glycosaminoglycans and counterstaining with fast green. Osteoid matrix and bone marrow are visible. Scale bars = 200 µm. (B): Stainings for metalloproteinase (MMP)13 and MMP9, as well as for the N-terminal neoepitope at the major MMP cleavage site (DIPEN) after 12 weeks in vivo (red/pink = positive). Scale bars = 50 µm. +, osteoid matrix; ⋆, bone marrow. Abbreviations: ALU, Arthrobacter luteus; H&E, hematoxylin and eosin; Masson, Masson’s trichrome; MMP, metalloproteinase; Saf-O, Safranin-O; SVF, stromal vascular fraction.
Ectopic bone formation. Grafts based on devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction cells from adipose tissue and implanted subcutaneously in nude mice. (A): Representative hematoxylin and eosin, Masson-Tri-Chrome, and Safranin-O (Saf-O) staining and in situ hybridization for human ALU sequences (dark blue = positive) after 12 weeks in vivo. Saf-O stainings are blue-green because of lack of glycosaminoglycans and counterstaining with fast green. Osteoid matrix and bone marrow are visible. Scale bars = 200 µm. (B): Stainings for metalloproteinase (MMP)13 and MMP9, as well as for the N-terminal neoepitope at the major MMP cleavage site (DIPEN) after 12 weeks in vivo (red/pink = positive). Scale bars = 50 µm. +, osteoid matrix; ⋆, bone marrow. Abbreviations: ALU, Arthrobacter luteus; H&E, hematoxylin and eosin; Masson, Masson’s trichrome; MMP, metalloproteinase; Saf-O, Safranin-O; SVF, stromal vascular fraction.

In the second experiment, Todorov and Kreutz used these SVF-seeded cartilage plugs to repair skull lesions in rats. Once again, the quantity of bone produced was directly proportional to the number of SVFs seeded into the cartilage matrices prior to implantation. In both experiments, the density of SVF cells positively correlates with the bone-forming cells in the grafts and the percentage of SVF-derived blood vessel-forming cells correlates with the amount of deposited mineralized matrix.

Bone repair capacity. Devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction (SVF) cells from adipose tissue and implanted in rat calvarial defects. (A): Mineralized volume quantified by microtomography (n = 9 grafts assessed per group). (B): Bone area assessed in histological sections, expressed as percentage of total defect area (n = at least 24 sections assessed per group). ∗∗∗∗, p < .0001. (C, D): Representative three-dimensional microtomography reconstructions (left) and hematoxylin/eosin (H&E) staining (right) of the calvarial defects filled with devitalized grafts, implanted without (C) or with (D) activation by SVF cells after 4 weeks. Dotted circles indicate the defect borders (4 mm diameter). Scale bars = 500 µm. (E): Microtomography (left) and H&E staining (middle and right) displaying the bridging between hypertrophic matrix and bone of the calvarium, or the fusion of single pellets (right) in activated grafts. White bar = 850 µm; black bars = 500 µm. Dotted lines indicate the edge of the calvarium. (F): In situ hybridization for Arthrobacter luteus sequences showing the presence of human cells (dark blue, positive) in the explants. Scale bar = 200 µm. Abbreviations: C, calvarium; dev, fibrin gel without stromal vascular fraction; dev + SVF, fibrin gel with stromal vascular fraction; P, hypertrophic matrix; SVF, stromal vascular fraction.
Bone repair capacity. Devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction (SVF) cells from adipose tissue and implanted in rat calvarial defects. (A): Mineralized volume quantified by microtomography (n = 9 grafts assessed per group). (B): Bone area assessed in histological sections, expressed as percentage of total defect area (n = at least 24 sections assessed per group). ∗∗∗∗, p < .0001. (C, D): Representative three-dimensional microtomography reconstructions (left) and hematoxylin/eosin (H&E) staining (right) of the calvarial defects filled with devitalized grafts, implanted without (C) or with (D) activation by SVF cells after 4 weeks. Dotted circles indicate the defect borders (4 mm diameter). Scale bars = 500 µm. (E): Microtomography (left) and H&E staining (middle and right) displaying the bridging between hypertrophic matrix and bone of the calvarium, or the fusion of single pellets (right) in activated grafts. White bar = 850 µm; black bars = 500 µm. Dotted lines indicate the edge of the calvarium. (F): In situ hybridization for Arthrobacter luteus sequences showing the presence of human cells (dark blue, positive) in the explants. Scale bar = 200 µm. Abbreviations: C, calvarium; dev, fibrin gel without stromal vascular fraction; dev + SVF, fibrin gel with stromal vascular fraction; P, hypertrophic matrix; SVF, stromal vascular fraction.

This is not the first time scientists have proposed the use of cartilage plugs to induce the formation of new bone. Van der Stok and others and Bahney and colleagues were able to repair segmental bone defects in laboratory rodents. Is this technique transferable to human patients? Possibly. Hypertrophic cartilage is relatively easy to make and it is completely conceivable that hypertrophic cartilage wedges can be sold as “off-the-shelf” products for bone treatments. SVF can also be derived from the patient or can be derived from donors.

Furthermore, the protocols in this paper all used human cells and grew the products in immunodeficient rats and mice. Therefore, in addition to scaling this process up, we have a potentially useful protocol that might very well be adaptable to the clinic.

The efficacy of this technique must be confirmed in larger animal model system before human trials can be considered. Hopefully human trials are in the future for this fascinating technique.

Fat-Based Mesenchymal Stem Cell-Seeded Matrix Heals Bronchopleural Fistula in Female Cancer Patient


Bronchopleural fistulae, mercifully abbreviated as BPF, refers to an opening or hole in the respiratory tree that causes continuity between the pleural space that surrounds the lungs and the bronchial tree. BPH is a highly feared complication of surgery on the respiratory system.

BPH can complicate surgical resection of the pulmonary system. Patients with lung cancers may require lung resection in order to remove tumorous lung tissue. The rate of BPH incidence after lung surgery varies widely, with reported incidences ranging from 1.5 to 28%. Necrosis or death of lung tissue as a result of infection can also cause BPH, as can tuberculosis. Chemotherapy or radiation therapy for lung cancers can also result in BPF. Finally, BPF may caused by persistent spontaneous pneumothorax, which refers to an abnormal build up of air or other gases in the pleural space, which causes an uncoupling of the lung from the chest wall.

To date, treatment for BPF is only partially effectively. The main treatment includes surgery, but the rate of recurrence of the fistulae remains rather high as do the rate of mortality. Can stem cells show us a better way?

Perhaps they can. Dennis A. Wigle, a surgeon at Mayo Clinic, and his collaborators used a synthetic bioabsorbable matrix seeded with the patients one fat-based mesenchymal stem cells to heal a BPF in a 63-yr old woman. Mind you, this is a case study (the lowest quality clinical evidence) and not a controlled study,. However, the success of this case study is at least suggestive that such an approach might prove useful for patients who suffer from BPFs.

Microscopic assessment of matrix cell seeding. (A): Ethidium bromide (red) and Syto-13 (green) costain demonstrating live and dead cells on mesenchymal stem cell seeding on matrix. (B): Confocal microscopy with CD90 (Thy-1) fluorescein isothiocyanate (green) and Hoechst 33342 (trihydrochloride trihydrate) (blue) fluorescent nuclear staining. These images were captured using a ×20 objective and a ×10 eyepiece, for a combined magnification of ×200. Scale bar = 150 µm.
Microscopic assessment of matrix cell seeding. (A): Ethidium bromide (red) and Syto-13 (green) costain demonstrating live and dead cells on mesenchymal stem cell seeding on matrix. (B): Confocal microscopy with CD90 (Thy-1) fluorescein isothiocyanate (green) and Hoechst 33342 (trihydrochloride trihydrate) (blue) fluorescent nuclear staining. These images were captured using a ×20 objective and a ×10 eyepiece, for a combined magnification of ×200. Scale bar = 150 µm.

A 63-yr old woman who had surgical resection of the lung in order to treat her lung cancer had, as a consequence of her surgery, a BPF. Some 30 different surgical attempts were made to repair the BPF, but all of them failed. The woman’s health declined and her medical team started to think of alternative treatments.

Fortunately, Mayo Clinic has been participating in an ongoing clinical trial to use fat-based mesenchymal stem cells to treat anal fistulae in Crohn’s disease patients. Therefore Dr. Wigle and his team considered using the protocol utilized with Crohn’s patients to repair this woman’s BPF.

Fat biopsies were taken from the patient and the fat was washed, minced, digested with enzymes, and then grown in special culture media. The adipose tissue-derived mesenchymal stem cells (AD-MSCs) grew and were isolated, characterized and shown to be MSCs.

These cells were then seeded on a matrix of synthetic bioabsorbable poly(glycolide-trimethylene carbonate) copolymer and then placed in a bioreactor to grow. After about 4 days, the matrix was flush with AD-MSCs, and this cell-seeded patch was then used in a subsequent surgery to seal the opening in the respiratory tree. This time the surgery worked. The patient was discharged 25 days after the surgery and sent home.

MRIs of the respiratory system showed that the BPF had indeed closed and properly resolved.

Preoperative imaging showing size and location of fistula, and postoperative imaging demonstrating disease resolution. (A): Preoperative bronchoscopy demonstrating large bronchopleural fistula (BPF) cavity and lateral extension of fistula tracts. (B): Postoperative bronchoscopy (3 months) demonstrating progressive healing of BPF site. (C): Preoperative computed tomography scan demonstrating large BPF with connection to atmosphere (additional axial slices inferiorly). (D): Postoperative computed tomography scan (16 months) demonstrating resolution of BPF.
Preoperative imaging showing size and location of fistula, and postoperative imaging demonstrating disease resolution. (A): Preoperative bronchoscopy demonstrating large bronchopleural fistula (BPF) cavity and lateral extension of fistula tracts. (B): Postoperative bronchoscopy (3 months) demonstrating progressive healing of BPF site. (C): Preoperative computed tomography scan demonstrating large BPF with connection to atmosphere (additional axial slices inferiorly). (D): Postoperative computed tomography scan (16 months) demonstrating resolution of BPF.

This case study might confirm what was previously observed in large animal studies by Petrella and others, namely that AD-MSCs can be used to heal BPF. Petrella and others theorized that implanted MSCs induce the proliferation of fibroblasts that then deposit collagen, which seals the BPF (see Ann Thorac Surg 97:480483.  Alternatively, AD-MSCs might differentiate into cell types  required for regeneration of the airways (Dominici M, and others, Cytotherapy 8:315317).  Either way, this paper seems to suggest that AD-MSCs can be isolated from a patient’s fat (even a very sick patient like this one) without incident and used for tissue engineering applications that can repair very serious wound like BPF. 

This paper was published in: Johnathon M., Aho, et Al., “Closure of a Recurrent Bronchopleural Fistula Using a Matrix Seeded With Patient-Derived Mesenchymal Stem Cells.” Stem Cells Trans Med October 2016 vol. 5 no. 10 1375-1379. 

New Stem Cell Treatment for Bronchopleural Fistulas


Mayo Clinic researchers have made history by using a patient’s own stem cells to heal an open wound on the upper chest of a patient that had been caused by postoperative complications of lung removal.

A hole in the chest that opens to the outside is called a bronchopleural fistulae. Such wounds are holes that lead from large airways in the lungs to the membrane that lines the lungs.

Unfortunately, present treatments for bronchopulmonary fistulae tend to be terribly successful and death from such injuries are all too common.

According to Dr. Dennis Wigle, a Mayo Clinch Researcher, “Current management is not reliably successful. After exhausting therapeutic options, and with declining health of the patient, we moved toward a new approach. The protocol and approach were based on an ongoing trial investigating this method to treat anal fistulas in Cohn’s disease”.

So Dr. Wigle and his colleagues harvested stem cells from the belly fat of their patient and seeded onto a bioabsorbable mesh that was surgically implanted at the site of the fistula.

Follow-up imaging of the patient showed that the fistula had closed and remained healed. More than a year-and-a-half later, the patient remains asymptomatic and has been able to resume activities of daily living.

In their paper, Wigle and others describe their patient, a 63-year-old female patient, who was referred to Mayo Clinic for treatment of a large bronchopleural fistula.

Because present therapies offer little relief, Wigle and his team turned to regenerative therapies in order to try a more innovative treatment.

“To our knowledge, this case represents the first in human report of surgically placed stem cells to repair a large, multiple recurrent bronchopleural fistula. The approach was well tolerated suggesting the potential for expanded use,” said Dr. Wigle.

While this procedure was successful in this case, it is unclear if this treatment was the main contributor to the healing of the wound. Since this is a single-patient case study and not a double blinded, placebo-controlled study, it is lower-quality evidence.

However, Wigle and others hope to further examine this technique, and in particular, the use a patient’s own stem cells, to treat fistulae in the respiratory system.

This case study was published in Stem Cells Translational Medicine, June 2016 DOI:10.5966/sctm.2016-0078.

Phase I Clinical Trial of Fat-Based Mesenchymal Stem Cells for Severe Osteoarthritis


In the July 2016 edition of the journal Stem Cells Translational Medicine, a report has been published that lays out the results of a phase I clinical trial that used mesenchymal stem cells from a patient’s own fat tissues to treat osteoarthritis of the knee.  This study was not placebo controlled, but did examine the effects of escalated doses on the patient.  The main  investigator for this trial was Dr. Christian Jorgensen from Lapeyronie University Hospital in Montpellier, France.

Osteoarthritis (OA) is the most common musculoskeletal condition in adults and it can cause a good deal of pain and disability.

Joints like the knee consist of a junction between two or more bones.  The ends of these bones are capped by layer of cartilage called “hyaline cartilage” that serves as a shock absorber.  Larger joints like the knee, shoulder, and hip are encased in a sac called the “bursa” that is filled with lubricating synovial fluid.

Knee

OA involves damage and/or destruction of the cartilage caps at the ends of long bones, and erosion and ultimately permanent changes in the structure of bone that underlies the cartilage at the end of the bone. The knee loses its shock absorbers and lubricators and becomes a grinding, inflamed, painful caricature of its former self.

To treat OA, most orthopedic surgeons will replace the damaged knee with an artificial knee that is attached the upper (femur) and lower (tibia and fibula) bones of the leg.  This procedure, arthroplasty, reconstructs the knee with artificial materials that form synthetic joints.  Alternatively, some enterprising physicians have tried to use stem cells from bone marrow to repair eroded cartilage in the knees of OA patients.  Christopher Centeno and his colleagues at his clinic near Denver, CO and affiliated sites have pioneered procedures for OA patients.  However, Dr. Centeno remains skeptical of the ability of stem cells from fat to treat patients with OA.

In animal studies, OA of the knee can be induced by injected tissue-destroying enzymes.  If laboratory mice that received injectionof these enzymes into their knees are then treated with fat-based mesenchymal stem cells, the effects and symptoms of OA do not appear (ter Huurne M, et al. Arthritis Rheum 2012; 64:3604-3613).  In another study in rabbits, injections of 2-6 million fat-derived mesenchymal stem cells into the knee-joint of rabbits suffering from OA improved cartilage health and inhibited cartilage degradation.  These administered cells also reduced inflammation in the knee (Desando G., et al., Arthritis Res Ther 2013; 15:R22).  Therefore, fat-based mesenchymal stem seem to have some ability to ameliorate the effects and consequences of OA, at least in preclinical studies.  This trial is the beginnings of what will hopefully be a series of experiments that will assess the ability (or inability) to treat OA patients.

18 patients were enrolled from an initial pool of 48 candidates who all suffered from severe, symptomatic OA of the knee.  Six patients received 2 million mesenchymal stem cells isolated from their own fat, 6 others received ten million mesenchymal stem cells isolated from their own fat, and the final group of 6 OA patients received 50 million mesenchymal stem cells isolated from their own fat tissues.  These mesenchymal stem cells were isolated from the patient’s fat that was collected by means of liposuction.  The fat was then processed by means of a standard protocol that is used to isolated mesenchymal stem cells from human fat (see Bura A, et al., Cytotherapy 2014; 16:245-257).  All patients received their stem cells by means of injection into the knee-joint (inter-articular injections).

Because this is a Phase I clinical trial, assessing the safety of the procedure is one of the main goals of this study.  No adverse effects were associated with either the liposuction or the interarticular injections.  The article even states: “Laboratory tests, vital signs and electrocardiograms indicated no local or systemic safety concerns.”. Four patients experienced slight knee pain and joint effusion that either resolved by itself or with treatment with a nonsteroidal antinflammatory drug (think ibuprofen).  Therefore it seems fair to conclude that this procedure seems safe, but a larger, placebo-controlled study is still required to confirm this.

As to the patient’s clinical outcomes, 17 of the 18 patients elected to forego total knee replacement.  All patients showed improvement in pain and knee functionality at 1 week, 3 months and 6 months after the procedure.  However, only the low-dose group showed improvements that were statistically significant.

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WOMAC pain and function improvement during the study (WOMAC = Western Ontario and McMaster Universities Arthritis Index)

WOMAC pain and function improvement during the study. Abbreviation: WOMAC, Western Ontario and McMaster Universities Arthritis Index.

Seven of the patients treated in Germany (11 patients were treated in France and 7 were treated in Germany) were also examined with Magnetic Resonance Imaging (MRI) before and 4 months after the procedure.  Six of the seven patients showed what could be interpreted as improvements in cartilage.

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dGEMRIC and T1rho magnetic resonance imaging (MRI) of selected patients. The graphs on the left show the dGEMRIC (n = 6) and T1rho (n = 5) values before and 4 months after cell therapy. Increasing dGEmRIC and decreasing T1rho values are each known to correspond to increasing glycosaminoglycan/proteoglycan content and thus improved cartilage condition. On the right, the corresponding dGEMRIC and T1rho maps are shown as a color-coded overlay on an anatomical MRI for a patient receiving a low cell dose. The observed values in the cartilage change in the time course can be easily seen and correspond to an increase in cartilage condition. Abbreviation: dGEMRIC, delayed gadolinium-enhanced magnetic resonance imaging of cartilage.

Tissue biopsies of 11 of the 18 patients revealed an absence of significant inflammation, but some patients (4-5) showed signs of weak or moderate inflammation.  One patient showed what seemed to be a sheet of MSC cells on the surface of the cartilage.

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Histologic findings. (A): Vascular congestion and weak lymphocytic infiltrate of the synovial (case 8) (magnification, ×50). (B): Osteoarthritic cartilage OARSI grade >3 (case 4) (×25). (C): Toluidine blue staining (case 2) (magnification, ×100). (D): Stem cell stroma shows an Alcian blue depleted matrix compared with the strong staining of osteoarthritic cartilage (case 2) (magnification, ×100). (E): Weak PS100 staining of possible stem cells on the cartilage surface and strong PS100 staining of chondrocytes (case 2) (magnification, ×100). Abbreviations: OARSI, Osteoarthritis Research Society International.

The primary outcome of this study – the safety of interarticular injections of fat0-based mesenchymal stem cells – seems to have been satisfied.  This is similar to the safety profiles of such cells in clinical trials that have used fat-based mesenchymal stem cells to treat fistulae in inflammatory bowel disease (Bura A, et al., Cytotherapy 2014; 16: 245-257) or critical limb ischemia (Lee WY and others, Stem Cells 2013; 31:2575-2581).  Also, patients showed improvements in pain and functionality.  Even though there was no placebo in this study, a double-blinded, placebo-controlled study that examined the use of efficacy of interarticular hyaluronic acid injections showed a smaller decreased in pain score that what was observed in this case (22.9 ± 1.4 vs 30.7 ± 10.7).  It is doubtful that the injected mesenchymal stem cells made much cartilage but instead quelled inflammation and stimulated resident stem cell populations to repair damage in the knee.

This study is small and is not placebo controlled, however, the hopeful results do warrant a larger, phase 1/2 placebo-controlled study that is apparently already underway.

An even more intriguing project might be to prime the isolated mesenchymal stem cells to make cartilage and then use live fluoroscopy to overlay the cells on the actual cartilage lesions.  While this is a more exacting procedure, it is the way Centeno and his group are using stem cells to treat their patients, and a true head-to-head study of the efficacy of fat-based mesenchymal stem cells versus bone marrow-based mesenchymal stem cells would be immensely useful.

Fat-Derived Stem Cells Form Muscle in Muscular Dystrophy Mice


Stem cell therapy for Duchenne muscular dystrophy (DMD) has been plagued by poor cell engraftment into diseased muscles. Additionally, there are no reports to date describing the efficient generation of muscle progenitors from fat-derived stem cells (ADSCs) that can contribute to muscle regeneration.

A study by Cheng Zhang and others from Sun Yat-sen University in Guangzhou, China, Guangdong Province has examined the ability of progenitor cells differentiated from ADSCs using forskolin, basic fibroblast growth factor, the glycogen synthase kinase 3β inhibitor 6-bromoindirubin-3′-oxime as well as the supernatant of ADSC cultures to form workable muscle cells.

When these fat-derived stem cells were treated as described above, they formed a proliferative population of muscle progenitors from ADSCs that had characteristics similar to muscle satellite cells. Furthermore, in culture, these cells were capable of terminal differentiation into multinucleated myotubes.

When these fat-derived stem cells were transplanted into mice that had an inherited type of DMD, the progenitor cells successfully engrafted in skeletal muscle for up to 12 weeks, and generated new muscle fibers, restored dystrophin expression, and contributed to the satellite cell compartment.

These findings highlight the potential application of ADSCs for the treatment of muscular dystrophy. They also illustrate the ability of ADSCs to differentiate into functional skeletal muscle cells when treated properly in culture. These same cells might serve as a treatment for DMD patients.

This article was published in Hum. Mol. Genet. (2015) doi: 10.1093/hmg/ddv316.

Fat-Based Stem Cells Speed the Healing of Bed Sores in Animals


Pressure ulcers, which are also knows as bedsores (or decubitus ulcers) are localized injuries to the skin that can also include the underlying tissue that usually occur as a result of pressure, or pressure in combination with rubbing or friction. They tend to occur some sort of bony prominence such as elbows, hips, shoulders, ankles, back of the head, and other such places. More than 2.5 million patients each year in the U.S. require treatment for pressure ulcers, and the elderly are at particularly high risk for these lesions. Currently, therapies for pressure ulcers consist of conservative medical management for shallow lesions and aggressive debridement and surgery for deeper lesions.

Jeffery Gimble and his colleagues from the Tulane University School of Medicine in New Orleans, Louisiana, used a mouse model for pressure ulcers to test the ability of fat-derived stromal/stem cell treatment to accelerate and enhance the healing of pressure ulcers.  The dorsal skin of both young (2 months old) and old (20 months old) C57BL/6J female mice was pinched between external magnets for 12 hours over 2 consecutive days. This treatment initiated a pressure ulcer, and one day after induction of the pressure ulcers, some of these mice were injected with fat-derived stromal stem cells that had been isolated from healthy mice that were of the same genetic lineage as the injured mice. However, the donor mice were genetically engineered to express a green fluorescent protein in all their tissues. Other mice were treated with injections of saline-treated controls.

The mice that were injected with fat-derived stromal/stem cells displayed a cell-concentration-dependent acceleration of wound closure. The cell-injected mice also showed improved epidermal/dermal architecture, increased fat deposition, and reduced inflammation at the sites of injury. Interestingly, these fat-derived stem cell-induced improvements occurred in both young and elderly mice. However, the gene expression profile of genes involved in the making of blood vessels, regulating the immune system, and tissue repair differed according to the age of the mice, with younger mice making more of these genes that their older counterparts. These results are consistent with clinical reports of the improved skin architecture after fat grafting in patients with thermal injuries.

This current proof-of-principle study sets the stage for clinical translation of the transplantation of fat-based stem cells as a treatment of pressure ulcers.

A Patient’s Own Stem Cells Treats Their Crohn’s Disease


Stem cells isolated from the fat of patients with Crohn’s disease, an inflammatory disease of the bowel, relieved them from fistulas, which are a common, and potentially dangerous side effect of the disease. This is according to the results of a phase 2 clinical trial published in the latest issue of STEM CELLS Translational Medicine (SCTM).

Patients with Crohn’s disease suffer from a painful, chronic disease in which the body’s immune system attacks its own gastrointestinal tract. In Crohn’s patients, inflammation within the bowel can sometimes extend completely through the intestinal wall and create a what is known as a “fistula.”. Fistulas are abnormal connections between the intestine and another organ or even the skin. If left untreated, a fistula can become infected and form an abscess that can be life threatening.

Chang Sik Yu, M.D., Ph.D., of the Asan Medical Center in Seoul, Korea, who is a senior author of the SCTM paper, describes the results of a clinical trial that was conducted in collaboration with four other hospitals in South Korea. According to Dr. Yu: “Crohn’s fistula is one of the most distressing diseases as it decreases patient’s quality of life and frequently recurs. It has been reported to occur in up to 38 percent of Crohn’s patients and over the course of the disease, 10 to 18 percent of them must undergo a proctectomy, which is a surgical procedure to remove the rectum.”

Overall, the treatments currently available for Crohn’s fistula remain unsatisfactory because they fail to achieve complete closure, lower recurrence of the fistulas and do not limit adverse effects, Dr. Yu said. Given the challenges and unmet medical needs in Crohn’s fistula, attention has turned to stem cell therapy as a possible treatment.

Several studies, including those undertaken by Dr. Yu’s team, have shown that mesenchymal stem cells (MSCs) do indeed improve Crohn’s disease and Crohn’s fistula. Their phase II trial enrolled 43 patients for a term of one year, over the period from January 2010 to August 2012. These patients received injections of their own fat-based MSCs, and 82 percent of them experienced complete closure of fistula eight weeks after the final ASC injection. 75 percent of the trial participants remained fistula-free two years later.

“It strongly demonstrated MSCs derived from ASCs are a safe and useful therapeutic tool for the treatment of Crohn’s fistula,” Dr. Yu said.

The latest study was intended to evaluate the long-term outcome by following 41 of the original 43 patients for yet another year. Dr. Yu reported, “Our long-term follow-up found that one or two doses of autologous ASC therapy achieved complete closure of the fistulas in 75 percent of the patients at 24 months, and sustainable safety and efficacy of initial response in 83 percent. No adverse events related to ASC administration were observed. Furthermore, complete closure after initial treatment was well sustained.”

“These results strongly suggest that autologous ASCs may be a novel treatment option for Crohn’s fistulae,” he said.

“Stem cells derived from fat tissue are known to regulate the immune response, which may explain these successful long-term results treating Crohn’s fistulae with a high risk of recurrence,” said Anthony Atala, M.D., Editor-in-Chief of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine.

ALK2 Manipulation Increases Bone Formation in Fat-Based Stem Cells


Mesenchymal stem cells possess a cell-surface protein called ALK2. ALK2 acts as a receptor for bone-inducing growth factors. ALK2, for example, is expressed in cartilage and if mesenchymal stem cells express a constantly-active form of ALK2, known as caALK2, these cells are driven to become cartilage-making cells (known as chondrocytes).

Can this receptor be used to drive bone formation? It turns out that manipulating ALK2 can drive fat-based stem cells (ASCs) to become bone making cells that ultimately improve bone tissue engineering. Researchers from the laboratory of Benjamin Levi at Massachusetts General Hospital, Boston, Massachusetts have fiddled with ALK2 in mesenchymal stem cells to for formation of bone from ASCs, and to enhance bone regeneration in a living animal.

To do this, Levi’s team genetically manipulated mice so that they expressed a form of ALK2 that was constantly turned on known as caALK2. The fat-based MSCs were then isolated and analyzed for their ability to make bone in culture. caALK ASCs were much more responsive to bone-inducing growth factors. These cells also expressed a whole host of bone-specific genes (e.g., Alp, Runx2, Ocn, Ops) after seven days. Since the caALK2 MSCs did so well in culture, they were then tested in mice with skull defects. Bone formation was significantly higher in mice treated with caALK2-expressing ASCs than those treated with normal ASCs.

Thus, Levi’s laboratory has shown that by treating mice with fat-based stem cells that express a constitutively active ALK2 receptor showed significantly increased bone formation. This increased bone formation can also be harnessed to improve skull healing in mice with bone defects.

Mesenchymal Stem Cells Assist Kidney Transplants in Cats


Dr. Chad Schmiedt, a veterinary surgeon from the University of Georgia (UGA) Veterinary Teaching Hospital, and his colleagues have used mesenchymal stem cells from the fat of cats to optimize the acceptance of a new kidney in cats.

The recipient of this kidney transplant was a four-year-old flame point Siamese male cat named Arthur. Arthur’s owners brought him from Virginia to the University of Georgia after he was diagnosed with chronic renal failure about a year ago. Two other veterinary hospitals declined to operate on Arthur, since they did not deem this cat an optimal candidate for a kidney transplant. As it turns out, Arthur has trouble absorbing cyclosporine, which is the anti-rejection drug used to prevent the recipient of the kidney transplant from rejecting it.

Arthur
Arthur

In his initial consultation with Arthur’s owners, Schmiedt had the idea of using adult feline stem cells as a part of Arthur’s immunosuppressive protocol. There was precedent for this, since a cat that was operated on at University of Georgia Veterinary Teaching Hospital in 2013 had received a kidney transplant with doses of its own mesenchymal stem cells to prevent rejection of the transplanted organ. This cat was doing well one year after surgery.

“To the best of my knowledge, UGA is the only veterinary facility in the world to use adult stem cells in feline kidney transplantation,” said Schmiedt, who actually heads UGA’s feline kidney transplant program.”

Schmiedt continued: “We used feline adult stem cells in one other transplant that we did last year. A study published in 2012 found that the use of MSCs during renal transplant surgery i humans lowered the risk of acute organ rejection, decreased the risk of infection, and the patients had better estimated renal function one year after surgery.”

Mesenchymal stem cells can be harvested fat, bone marrow, and umbilical cord or placenta. Before the transplant surgery, Schmiedt isolated mesenchymal stem cells (MSCs) from Arthur’s fat and the UGA Regenerative Medicine Service grew the stem cells from the fat sample for use in Arthur after his surgery.

Arthur has his kidney transplant on May 15, 2014. The first surgery harvests a kidney from the donor cat (named Joey) and the second surgery transplants the donated kidney into Arthur. The UGA transplant program for cats requires that the donor cat be adopted by the recipient family’s family, which means that Joey and Arthur will become lifelong playmates.

“Cat owners who seek kidney transplants for their sick cats have to be very dedicated,” said Schmiedt. “They will give their car medication twice a day for the rest of its life. They also must be willing to take their cats to the veterinarian for frequent check-ups… a significant amount of time and expense is involved in keeping the recipient and donor cats healthy. But cat lovers who will go to this extent are willing to extend this kind of care to all cats they own.”

Apparently, Joey will be joined by Arthur and five other felines as well.

Stem cells do not replace the need for antirejection medication, and since Arthur’s body poorly absorbs cyclosporine, he will need to take a second antirejection drug as well called mycophenolate. Schmiedt, however, and his colleague stem cell scientist Dr. John Peroni sees MSCs making an important contribution to transplant medicine: “MSCs in veterinary species have been primarily used to treat musculo-skeletal injury – problems with bones, tendons, and joints – and those are our most frequent uses here at the UGA College of Veterinary Medicine. But there is good evidence to support using stem cells to modulate the immune system and regulate inflammation. So, the transplant setting might be another optimal use for these types of stem cells.”

In order to access the efficacy of MSCs in a transplant setting, controlled studies must be done. It is clear that transplanted MSCs do not improve kidney function, but they do seem to slow down the progression of kidney disease. Schmiedt thinks that benefits to patients are possible: “The only down side is harvesting the cells seven to 10 days ahead of the surgery, which adds to the cost of transplant procedure.”

Adaptation of this procedure to animals could smooth the path to making this procedure readily available in humans as well.

The Australian Football League Approves Regeneus’ Fat-Based HiQCell Stem Cell Therapy for Injured Players


The regenerative medicine company Regeneus Ltd announced this week that the Australian Football League or AFL has decided to approve, on a case-by-case basis, the use of its innovative HiQCell stem cell therapy as an optional treatment for injured AFL players. Football (soccer) players tend to suffer from impact-related osteoarthritis and tendonitis.

Regeneus’ Commercial Development Director for Human Health, Steve Barbera, said, “It’s pleasing that HiQCell has been approved under the new AFL Prohibited Treatments List released in March 2014. HiQCell also received clearance as an approved therapy from the Australian Sports Anti-Doping Authority (ASADA) for use with athletes who participate in sporting competitions subject to the WADA Anti-Doping Code, including the AFL. This recent decision by the AFL demonstrates a further level of compliance, specifically for players within that sporting code.”

Regeneus’ HiQCell treatment is the only stem cell treatment for osteoarthritis that has been subjected to the highest level of clinical scrutiny. A double-blind placebo-controlled safety trial is the gold standard for clinical trials. The particular clinical trial to which HiQCell treatments were subjected showed that HiQCell is safe and it reduces pain and halts cartilage degradation in arthritic joints. Additionally, the ongoing effects of HiQCell are being tracked in over 380 patients in an independent ethics-approved registry. A recent registry update demonstrated that patients are maintaining significant improvements 2 years after their treatment.

HiQCell has already been used to treat several high-profile athletes across several sporting codes, including the National Rugby League, which was announced on May 7th, 2014. It is encouraging for Regeneus that elite sports patients can use their HiQ therapy to much quickly return to sports from hard-to-treat injuries and continue their playing careers after receiving this innovative therapy.

Dr Phil Bloom, a Melbourne based Specialist Sports and Exercise Physician and HiQCell treating medical practitioner, said, “permission from the AFL for HiQCell treatment is a positive progression as it allows for an additional option for players with conditions that are unresponsive to existing treatments”.

The HiQCell treatment uses stem cells harvested from a small amount of a patient’s fat. After separating and concentrating these regenerative cells, they are re-injected in osteoarthritic-affected joints such as knees, hips and ankles. The HiQCell treatment reduces inflammation and repairs damaged tissue when it is carried out under the supervision of a medical practitioner.

Mesenchymal Stem Cells from Fat Relieve Arthritis Pain for Up to Two Years


Regeneus is an Australian regenerative that has developed an experimental treatment for arthritis called HiQCells.  HiQCell is a stem cell treatment made from the patient’s own adipose (fat) tissue, and is subsequently injected into an affected joint or tendon. Regeneus has tested their HiQCell treatment in an independent clinical study that examined the efficacy of injections of HiQCells into the joints of patients with osteoarthritis of the knee.  The study examined 40 patients with knee osteoarthritis.  Half of these patients received the placebo and half HiQCell in a double-blinded study.  When asked about their pain levels six months after the procedure, patients in the placebo and HiQCell group ~45% of patients reported less pain and by 12 months after treatment 55% of patients in both groups reported less pain.  Thus both treatments relieved pain to a similar degree.  However, when the progression of the disease was examined, a very different result was observed.  As osteoarthritis progresses, some of the breakdown products of joint cartilage appear in urine and blood.  By collecting urine and blood samples from osteoarthritis patients, the progression of the disease can be readily tracked.  Blood and urine testing showed significantly less cartilage breakdown in the HiQCell group and significantly more breakdown in the patients in the placebo group who had advanced cartilage damage. Thus, even though the patients who received placebo had about the same level of pain reduction over the six-month period, it seems that their cartilage breakdown progressed at a faster rate.

Now a follow-up examination of these and other subjects who participated in this initial clinical study has revealed something surprising.  According to Regeneus, as of July 21, 2014, from a collection of 386 patients: 1) Pain has continued to decrease two years post-treatment; 2) One year after treatment, 63 of 86 patients reported more than a 30% reduction in pain;
3) Two years after treatment, 14 of 17 patients reported more than a 30 % reduction in pain and 14 patients experienced an average pain reduction of 84% at two years post-treatment; 4) Patients also reported significant improvements from pre-treatment in knee-function, sleep quality and reduced pain medications.  Finally, it is clear from these results that HiQCell is a safe therapy and well tolerated by patients, since the frequency and severity of adverse effects of patients who received HiQCell treatments were no different from those received the placebo.

The HiQCell Joint Registry established by Regeneus is the first of its kind in that the patients who participate in this study are subjected to long-term follow-up and undergo stem cell therapy using the patient’s own fat-derived stem cells. The HiQCell study has been approved by a human research ethics committee.  These 386 patients included in the Joint Registry will continue to be followed for up to 5 years with analysis updated regularly.

Professor Graham Vesey, CEO of Regeneus, comments: “The registry data is demonstrating that HiQCell has a therapeutic benefit for longer than 2 years. We are now also beginning to see very encouraging data from patients that have had cells frozen for future injections. This combination of the long-term effect from HiQCell and the successful storage of cells for repeat injections in the future, means that HiQCell can be used to treat joint pain for many years. This is particularly important for patients that are too young for joint replacement or are simply looking to delay joint replacement.”

Fat-Based Stem Cells Support New Brain Cell Growth in Alzheimer’s Disease Mice


Alzheimer’s disease (AD) causes progressive death of brain cells and dementia. The loss of memory, coordination, and eventually motor function is relentless and horrific, and causes extensive suffering, financial pressures and loss. Stem cell treatments have been proposed as a treatment for AD, but such treatments have met resistance because of the complex pathology of AD. Introducing new neurons into the brain will do little good if cells are normally dying. However, some work with laboratory animals has suggested that stem cell treatments can benefit animals with conditions that approximately AD (see Kim S, et al., PLoS One. 2012;7(9):e45757; Bae JS, et al., Curr Alzheimer Res. 2013 Jun;10(5):524-31). However there are few studies that examine the therapeutic effect of mesenchymal stem cells from fat tissue or “adipose-derived stem cells” on mice with AD, and the effect of these cells on the oxidative injury that tends to accompany AD, and if these stem cells stimulate the generation of new neurons in the brains of AD mice.

Now we have evidence that transplantation of mesenchymal stem cells can stimulate for formation of new brain cells in adult rat or mouse models of AD and improve tissue structure and function after a stroke. Dr. Yufang Yan and her team from the School of Life Sciences at Tsinghua University, China transplanted adipose-derived stromal cells (ADSCs) into a part of the brain known as the hippocampus of mice that express the APP/PS1 transgene. Such mice show an AD-like disease, with memory loss and amyloid plaques that form in the brain.

Transplantation of ADSCs in these AD model mice decreased oxidative stress and promoted the growth of new neurons and glial cells in the subgranular and subventricular zones of the hippocampus, and, consequently improved the cognitive impairment in APP/PS1 transgenic AD mice.

These findings were published in Neural Regeneration Research (Vol. 9, No. 8, 2014), and provide theoretical and experimental evidence that ADSCs can be used to treat AD patients.

Heart Function Improved by Injecting Discarded Surgery Fat


Many patients with heart problems – such as heart disease or angina – may need to undergo cardiac surgery in order to restore or improve blood flow. But a new study suggests that the procedure may offer so much more; stem cells in fat discarded during cardiac surgery could be injected back into the patient’s heart to further improve its function.

A research team led by senior author Canadian cardiologist Dr. Ganghong Tian will present their findings at the Frontiers in Cardiovascular Biology meeting in Barcelona, Spain.

Previous work by this group has shown that subcutaneous fat (adipose tissue) contains stem cells that can reduce the severity of heart attacks, improve cardiac function, and augment blood vessel regeneration in laboratory animals with experimentally induced heart attacks. These fat-based stem cells can be easily obtained through liposuction. However, Tian noted, “But obtaining these from a patient undergoing cardiac surgery requires pre-surgery to collect adipose tissue from the subcutaneous region.”

Is there a better way? According to Tian, during cardiac surgery, the surgeon often removes fat tissue that resides around the heart (so-called mediastinal fat) in order to properly expose the heart. Tian wondered if this fat contain stem cells that could be re-introduced to the heart to improve its function after heart surgery

In order to test this hypothesis, Tian and others collected mediastinal fat tissue from 24 patients who had undergone cardiac surgery. Then Tian’s group injected rats with mediastinal fat stem cells. The rats injected with stem cells from mediastinal fat showed greater ventricular movement in their hearts and no reduction in left ventricular ejection fraction.

Closer examination of the stem cells from mediastinal fat showed that mediastinal fat housed a rather robust number of stem cells, and that these stem cells could differentiate into fat and bone cells. Also, these stem cells expressed genes that are often found in heart muscle cells.

With this pre-clinical information in hand, Tian and others examined the use of mediastinal fat-based stem cells in 13 rats with congestive heart failure. These stem cells were directly injected into the hearts of eight rats, and five were injected with a saline solution.

After 6 weeks, all the rats underwent magnetic resonance imaging (MRI). When the five control rats were compared with those who those rats that received injections of mediastinal fat-based stem cells, the stem cell-injected rats demonstrated greater ventricular movement in their hearts and no reduction in left ventricular ejection fraction (ejection fraction measures how much blood is being pumped out of the left ventricle of the heart).

Commenting on the team’s findings, Dr. Tian says: “This is the first evidence that stem cells collected from the mediastinal fat region are cardioprotective. They displayed the same cardioprotective capacity we found in our previous research on stem cells from subcutaneous fat tissue. This raises the exciting possibility of using a patient’s own stem cells, isolated from waste tissue during cardiac surgery, to improve their heart function.”

Tian noted that there are currently some issues with this procedure that need to be addressed with further research. Techniques must be developed to quickly isolate stem cells from mediastinal fat so they can be injected back into a patient’s heart during cardiac surgery. Tian said, “It currently takes several hours to purify the cells and we are looking for collaborators to help us devise a more efficient method.”

Tina and others would also like to examine the ability of these stem cells to improve cardiac function long-term, beyond the 6 weeks monitored in this study. Furthermore, Tian and his group would like to induce the stem cells into functional heart muscle cells that display electrical pulses and beating.

Cartilage Production From Fat-Based Stem Cells Without Exogenous Growth Factors


Making cartilage from fat-based stem cells would be so much more attractive if we didn’t have to use exogenous sources of growth factors. Nevertheless, fat-based stem cells remain quite attractive as a source of cartilage since these cells can be grown in culture to large numbers and can also be readily differentiated into chondrocytes if they are stimulated with the growth factor transforming growth factor-β1 (TGF-β1). Using exogenous TGF-β1, however, has side undesirable effects. Is there another way?

Maybe. A new study by Loran Solorio and Eben Alsberg at Case Western Reserve University has used a culture medium containing TGF-β1-loaded microspheres to make cartilage from fat-based stem cells in culture. This technique can make cartilage without any exogenous growth factors, since all growth factors required for cartilage production are found within the culture system.

In this study, Solorio and Alsberg used exogenous TGF-β1 to induce cartilage formation in fat-based stem cells that were grown in sheets. These sheets of cells made cartilage after 3 weeks. Once it was clear that their experimental system worked well, they used TGF-β1-loaded gelatin microspheres to deliver the growth factor. By tweaking the quantity of microspheres and the concentration of TGF-β1 required for this to work, Solorio and Alsberg showed that the use of TGF-β1-loaded microspheres could induce cartilage formation as well as exogenous TGF-β1. Staining for cartilage-specific molecules and detailed microscopic observation of the cartilage showed that it was indeed, good, solid cartilage.

This publication is the first demonstration of the self-assembly of fat-derived stem cells into high-density cell sheets capable of forming cartilage in the presence of TGF-β1-releasing microspheres. The incorporation of these microspheres might bypass the need for extended culture of the stem cells, potentially allowing stem cells sheets to be implanted more rapidly into defects to regenerate cartilage in a living organism.

Using Fat Stem Cells to Treat a Deadly Cancer


Johns Hopkins University researchers have reported the successful use of stem cells derived from human body fat to deliver biological treatments directly to the brains of mice suffering from the most common and aggressive form of brain tumor. Such treatments significantly extended the lives of these cancer-stricken animals.

These experiments offer proof-of-principle that such a technique would work in human patients after surgical removal of brain cancers called glioblastomas. This technique provides a way to find and destroy any remaining cancer cells in those areas of the brain that are difficult to reach. Glioblastoma cells represent a challenge for cancer treatments, since they are quite sprightly, and can migrate across the entire brain, hide out and establish new tumors. Consequently, the cure rates for glioblastoma are notoriously low.

In the mouse experiments conducted by the Johns Hopkins group, investigators used mesenchymal stromal cells (MSCs) from fat tissue. Fat-based MSCs have a mysterious ability to sniff out cancer and other damaged cells. After genetically modifying the MSCs so that they secreted a protein called bone morphogenetic protein 4 (BMP4), these MSCs were injected into the brains of mice that suffered from glioblastomas. BMP-4 is a small, secreted protein that plays essential regulatory roles in embryonic development, but also has a demonstrated tumor suppression function.

Study leader Alfredo Quinones-Hinojosa, M.D., a professor of neurosurgery, oncology and neuroscience at the Johns Hopkins University School of Medicine and his colleagues published the results of this experiment in the journal Clinical Cancer Research. According to their results, those mice that were treated with the BMP-4-secreting fat-based MSCs had significantly less tumor growth and spread. In general the cancers in these animals were less aggressive and had fewer migratory cancer cells compared to mice that didn’t get the treatment. Also, the stem cell-treated mice survived significantly longer (an average of 76 days, compared to 52 days in the untreated mice).

“These modified mesenchymal stem cells are like a Trojan horse, in that they successfully make it to the tumor without being detected and then release their therapeutic contents to attack the cancer cells.”

Standard treatments for glioblastoma include chemotherapy, radiation and surgery. Unfortunately, even a combination of all three rarely leads to more than 18 months of survival after diagnosis. Discovering new ways to seek and destroy straggling glioblastoma cells that other treatments can’t get is a long-sought goal, says Quinones-Hinojosa. However, he also cautions that years of additional studies are needed before human trials of fat-derived MSC therapies could begin.

Quinones-Hinojosa also treated brain cancer patients at Johns Hopkins Kimmel Cancer Center, and he and his co-workers were greatly encouraged that the genetically-engineered stem cells let loose into the brain in his experiments did not transform themselves into new tumors.

These latest findings build on research published in March 2013 by Quinones-Hinojosa and his team, which demonstrated that harvesting MSCs from fat was much less invasive and less expensive than getting them from bone marrow (PLoS One, March 2013).

Ideally, he says, if MSCs work as a cancer treatment, a patient with a glioblastoma would have some adipose tissue (fat) removed from any number of locations in the body a short time before surgery. Afterwards, these fat-derived MSCs would be isolated and manipulated in the laboratory so that they would secrete BMP4. Then, after surgeons removed whatever parts of the brain tumor they could get to, they would deposit these BMP-secreting cells into the brain in the hopes that they would seek out and destroy the left-over cancer cells.

 

A Protein from Fat-Based Stem Cells Prevents Light-Induced Damage to the Retina


Japanese researchers from Gifu Pharmaceutical University and Gifu University have reported that a type of protein found in stem cells taken from adipose (fat) tissue can reverse and prevent age-related, light-induced retinal damage in mice. These results may lead to treatments for patients faced with permanent vision loss.

According to the work done by these two research teams led by Drs. Hideaki Hara and Kazuhiro Tsuruma, a single injection of fat-derived stem cells (ASCs) reduced the retinal damage induced by light exposure in mice. This study also discovered that when fat-derived stem cells were grown in culture with retinal cells, the stem cells prevented the retinal cells from suffering damage after exposure to hydrogen peroxide and visible light both in the culture and in the retinas of live mice.

Additionally, Hara and Tsuruma and their colleagues discovered a protein in fat-derived stem cells called “progranulin.” This protein, progranulin, seems to play a central role in protecting other cells from suffering light-induced eye damage.

In the retina, which lies at the back of the eye, excessive light exposure causes degeneration of the photoreceptor cells that respond to light. Several studies have suggested that a long-term history of exposure to light might be an important factor in the onset of age-related macular degeneration. Photoreceptor loss is the primary cause of blindness in particular eye-specific degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.

“However, there are few effective therapeutic strategies for these diseases,” Hideaki Hara, Ph.D., R.Ph., and Kazuhiro Tsuruma, Ph.D., R.Ph.

“Recent studies have demonstrated that bone marrow-derived stem cells protect against central nervous system degeneration with limited results. Just like the bone marrow stem cells, ASCs also self-renew and have the ability to change, or differentiate, as they grow. But since they come from fat, they can be obtained more easily under local anesthesia and in large quantities.”

The fat tissue used in the study was taken from mice and processed in the laboratory to isolate the fat-based stem cells. Afterwards, those cells were tested with cultured mouse retinal cells, and they show a robust protective effect. These successes suggested to the team to test their theory on a live group of mice that had retinal damage after exposure to intense levels of light.

Five days after receiving injections of the fat-based stem cells, the animals were tested for photoreceptor degeneration and retinal dysfunction. The results showed the degeneration had been significantly inhibited.

“Progranulin was identified as a major secreted protein of ASCs, which showed protective effects against retinal damage in culture and in animal tests using mice,” Drs. Hara and Tsuruma said. “As such, it may be a potential target for the treatment of degenerative diseases of the retina such as age-related macular degeneration and retinitis pigmentosa. The ASCs reduced photoreceptor degeneration without engraftment, which is concordant with the results of previous studies using bone marrow stem cells.”

“This study, suggesting that the protein progranulin may play a pivotal role in protecting against retinal light-induced damage, points to the potential for new therapeutic approaches to degenerative diseases of the retina,” said, Anthony Atala, MD, editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine, where this work was published.

Human Fat Contains Multilineage Differentiating Stress Enduring Cells With Great Potential for Regenerative Medicine


A collaboration between American and Japanese scientists has discovered and characterized a new stem cell population from human fat that do not cause tumors and can differentiate into derivatives from ectoderm, mesoderm, and endoderm.

Multilineage Differentiating Stress-Enduring or Muse cells are found in bone marrow and the lower layers of the skin (dermis). Muse cells are a subpopulation of mesenchymal stem cells, and even express a few mesenchymal stem cell-specific genes (e.g., CD105, a cell-surface protein specific to mesenchymal stem cells). However, Muse cells also express cell surface proteins normally found in embryonic stem cells (e.g., stage-specific embryonic antigen-3, SSEA-3). Additionally, Muse cells have the ability to self-renew, and differentiate into cell types from all three embryonic germ layers, ectoderm (which forms skin and brain), mesoderm, (which forms muscle, bone, kidneys, gonads, heart, blood vessels, adrenal glands, and connective tissue), and endoderm (which forms the gastrointestinal tract and its associated tissues). Finally, Muse cells can home to damaged sites and spontaneously differentiate into tissue-specific cells as dictated by the microenvironment in which the cells find themselves.

A new publication by Fumitaka Ogura and others from Tohoku University Graduate School of Medicine in Sendai, Japan and Saleh Heneidi from the Medical College of Georgia (Augusta, Georgia), and Gregorio Chazenbalk from the David Geffen School of Medicine at UCLA has shown that Muse cells also exist in human fat.

The source of cells came from two places: commercially available fat tissue and freshly collected fat from human subjects, collected by means of liposuction. After growing these cells in culture, the mesenchymal stem cells and Muse cells grew steadily over the 3 weeks. Then the Dezawa research group used fluorescence-activated cell sorting (FACS) to isolate from all these cells those cells that express SSEA-3 on their cell surfaces.

FACS uses antibodies conjugated to dyes that can bind to specific cell proteins. Once the antibodies bind to cells, the cells are sluiced through a small orifice while they are illuminated by the laser. The laser activates the dyes if the cell fluoresces, one door opens and the other closes. The cell goes to one test tube. If the cell does not fluoresce, then the door stay shut and another door opens and the cell goes into a different test tube.  In this way, cells with a particular cell-surface protein are isolated from other cells that do not have that cell-surface protein.

Fluorescent-Activated Cell Sorting
Fluorescent-Activated Cell Sorting

In addition to expression SSEA-3, the fat-based Muse cells expressed other mesenchymal stem cell-specific cell-surface proteins (CD29, CD90), but they did not express proteins usually thought to be diagnostic for fat-based mesenchymal stem cells (MSCs) such as CD34 and CD146.  Muse cells also expressed pluripotency genes (Nanog, Oct3/4, PAR4, Sox2, and Tra-1-81).  The Muse cells grew in small clusters and some cell expressed ectodermal-specific genes (neurofilament, MAP2), others expressed mesodermal-specific genes (smooth muscle actin, NKX2) and endodermal-specific genes (alpha-fetoprotein, GATA6).  These data suggested that the cultured Muse cells were poised to form either ectoderm, mesodermal, or endodermal derivatives.

When transplanted into mice with non-functional immune systems, the Muse cells never formed any tumors or disrupted the normal structure of the nearly tissues.  When placed in differentiating media, fat-derived Muse cells differentiated into cells with neuron-like morphology that expressed neuron-specific genes (Tuj-1), liver cells, and fat.  When compared with Muse cells from bone marrow or skin, the fat-derived Muse cells were better at making bone, fat, and muscle, but not as good as bone marrow Muse cells at making neuronal cell types, but not as good at making glial cells.  Many of these assays were based on gene expression experiments and not more rigorous tests.  Therefore, the results of these experiments might be doubtful until they are corroborated by more rigorous experiments.

These cells are expandable and apparently rather safe to use.  More work needs to be done in order to fully understand the full regenerative capacity of these cells and protocols for handling them must also be developed.  However, hopefully pre-clinical experiments in rodents will give way to larger animal experiments.  If these are successful, then maybe human trials come next.  Here’s to hoping.

An Efficient Method for Converting Fat Cells to Liver Cells


I have a friend whose wife has systemic lupus erythematosis, and her liver has taken a beating as a result of this disease. She has never had a drop of alcohol for decades and yet she has a liver that looks like the liver of a 70-year-old alcoholic. The scarring of the liver as result of repeated damage and healing has seriously compromised her liver function. She is now a candidate for a liver transplant. Wouldn’t it be nice to simply give her liver cells to heal her liver?

This dream came a little closer to becoming reality in October of this year when scientists at Stanford University developed a fast and efficient way to convert fat cells isolated from routine liposuction into liver cells. Even though these experiments used mice, the stem cells were isolated from human liposuction procedures.

This experiment did not use embryonic stem cells or induced pluripotent stem cells to generate liver cells. Instead it used adult stem cells from fat.

Fat-based stem cells

The liver builds complex molecules, filters and breaks down waste products and toxic substances that might otherwise accumulate to dangerous concentrations.

The liver, unlike other organs, has a capacity to regenerate itself to a significant extent, but the liver’s regenerative abilities cannot overcome the consequences of acute liver poisoning, or chronic damage to the liver, as a result of hepatitis, alcoholism, or drug abuse.

For example, acetaminophen (Tylenol) is a popular pain-reliever, but abusing acetaminophen can badly damage the liver. About 500 people die each year from abuse of acetaminophen, and some 60,000 emergency-room visits and more than 25,000 hospitalizations annually are due to acetaminophen abuse. Other environmental toxins, such as poisonous mushrooms, contribute more cases of liver damage.

Fortunately, the fat-to-liver protocol is readily adaptable to human patients, according to Gary Peltz, professor of anesthesia and senior author of this study. The procedure takes about nine days, which is easily fast enough to treat someone suffering from acute liver poisoning, who might die within a few weeks without a liver transplant.

Some 6,300 liver transplants are performed annually in he United States, and approximately 16,000 patients are on the waiting list for a liver. Every year more than 1,400 people die before a suitable liver can be found for them.

Even though liver transplantations save the lives of patients, the procedure is complicated, not without risks, and even when successful, is fraught with after effects. The largest problem is the immunosuppressant drugs that live patients must take in order to prevent their immune system from rejecting the transplanted liver. Acute rejection is an ongoing risk in any solid organ transplant, and improvements in immunosuppressive therapy have reduced rejection rates and improved graft survival. However, acute rejection still develops in 25% to 50% of liver transplant patients treated with immunosuppressants. Chronic rejection is somewhat less frequent and is declining and occurs in approximately 4% of adult liver transplant patients.

Peltz said, “We believe our method will be transferable to the clinic, and because the new liver tissue is derived from a person’s own cells, we do not expect that immunosuppressants will be needed.”

Peltz also noted that fat-based stem cells do not normally differentiate into liver cells. However, in 2006, a Japanese laboratory developed a technique for converting fat-based stem cells into induced liver cells (called “i-Heps” for short). This method, however, is inefficient, takes 30 days, and relies on chemical stimulation. In short, this technique would not provide enough material to regenerate a liver.

The Stanford University group built upon the Japanese work and improved it. Peltz’s group used a spherical culture and were able to convert fat-bases stem cells into i-Heps in nine days and with 37% efficiency (the Japanese group only saw a 12% rate). Since the publication of their paper, Peltz said that workers in his laboratory have increased the efficiency to 50%.

Dan Xu, a postdoctoral scholar and the lead author of this study, adapted the spherical culture methodology from early embryonic-stem-cell literature. However, instead of growing on flat surfaces in a laboratory dish, the harvested fat cells are cultured in a liquid suspension in which they form spheroids. Peltz noted that the cells were much happier when they were grown in small spheres.

Once they had enough cells, Peltz and his co-workers injected them into immune-deficient laboratory mice that accept human grafts. These mice were bioengineered in 2007 as a result of a collaboration between Peltz and Toshihiko Nishimura from the Tokyo-based Central Institute for Experimental Animals. These mice had a viral thymidine kinase gene inserted into their genomes and when treated with the drug gancyclovir, the mice experienced extensive liver damage.

After gancyclovir treatment, Peltz and his coworkers injected 5 million i-Heps into the livers of these mice, using ultrasound-guided injection procedures, which is typically used for biopsies.

Four weeks later, the mice expressed human blood proteins and 10-20 percent of the mouse livers were repopulated with human liver cells. Blood tests also showed that the mouse livers, which were greatly damaged previous to the transplantation, were processing nitrogenous wastes properly. Structurally, the mouse livers contained human cells that made human bile ducts, and expressed mature human liver cells.

Other tests established that the i-Heps made from fat-based stem cells were more liver-like than i-Heps made from induced pluripotent stem cells.

Two months are injection of the i-Heps, there was no evidence of tumor formation.

Peltz said, “To be successful, we must regenerate about half of the damaged liver’s original cell count.” With the spherical culture, Peltz is able to produce close to one billion injectable i-Heps from 1 liter of liposuction aspirate. The cell replication that occurs after injection expands that number further to over 100 billion i-Heps.

If this is possible, then this procedure could potentially replace liver transplants. Stanford University’s Office of Technology Licensing has filed a patent on the use of spherical culture for hepatocyte (liver cell) induction. Peltz’s group is optimizing this culture and injection techniques,talking to the US Food and Drug Administration, and gearing up for safety tests on large animals. Barring setbacks, the new method could be ready for clinical trials within two to three years, according the estimations by Peltz.

Clinical Study Evaluates Healing of Knee Cartilage With Stem Cells


The biotechnology company InGeneron will test its patented Transpose RT system in a clinical study that examined the ability of regenerative cells from a patient’s own fat to enhance cartilage healing after knee surgery.

Qualified patients are being recruited through the Fondren Orthopedic Group in Houston. According to the American Orthopedic Society for Sports Medicine, over 4 million knee arthroscopies are performed worldwide each year. Damaged knee cartilage is very difficult to treat and can lead to chronic pain and long-term disability.

Robert Burke, who is serving as the principal investigator of this clinical study, is an orthopedic surgeon with the Fondren Orthopedic Group in Houston. Burke thinks that stem cells taken from a patient’s own fat may enhance cartilage healing. He studied adding patient-derived regenerative cells to the knee during arthroscopic surgery for particular patients, and compared them to patients who had arthroscopic surgery without added fat-derived stem cells.

Arthroscopic surgery is a common procedure is commonly used to treat damaged cartilage, and the patients who had received arthroscopic surgery were randomly chose to either receive fat-derived stem cells or not receive them. Burke, will then monitor these patients for the next 12 months after surgery to determine if the added cells improved cartilage healing.

According to Burke, “Articular cartilage, the smooth surface covering the joints at the ends of bones, has no good way of healing on its own. The body doesn’t create enough new cartilage of the same type to repair the damage.” Better treatments would use various techniques to help the body make new cartilage.

“Stem cells and other regenerative cells that we can obtain fat have the potential to do that,” said Burke. Such regenerative cells can divide and mature to form several types of cells and tissues. and are found in multiple places in the body. Fat that lies just below the skin is one of the easiest places to obtain stem cells.

The InGeneron Transpose RT System uses a small amount of fat, which is removed and processed to separate out the regenerative cells. The separated adipose tissue-derived mesenchymal stem cells are then immediately placed into the area of damaged cartilage during knee surgery. Once in the knee, these cells may divide to make new cartilage cells.

This kind of biological activity has been seen in laboratory studies and veterinary medicine. However, Burke’s study will be one of the first to test the technology in humans for treating cartilage damage. Like other types of stem cell-based therapies, the treatment is not currently licensed for human use in the United States but it is registered in Europe, Mexico, and other countries. Following the Texas Medical Board’s rules about the use of stem cells for treatment, this study is under the supervision of the research review board at Texas Orthopedic Hospital, where all of the patients will undergo surgeries.

This is a two-year study.

Stem Cells Derived from Fat Show Promise for Regenerative Medicine


A detailed review article in the June issue of Plastic and Reconstructive Surgery, the official medical journal of the American Society of Plastic Surgeons, has examined the safety and clinical efficacy of fat-derived stem cells. Stem cells from fat, also known as ACSs, are a promising source of cells for use in plastic surgery and regenerative medicine, according to this review, but there are still many questions that remain about them. Much more research is needed in order to completely establish the safety and effectiveness of ASC-based therapies in human patients. The review article was written by ASPS Member Surgeon Rod Rohrich, MD of University of Texas Southwestern Medical Center, Dallas, and his colleagues (Dr. Rohrich is Editor-in-Chief of Plastic and Reconstructive Surgery).

ASCs are very easily procured from humans, since simple procedures such as liposuction can provide more than enough material for therapies. On the average, one gram of fat yields about 5,000 stem cells, whereas the yield from the same quantity of bone marrow is about 1,000 cells (B. M. Strem, K. C. Hicok, M. Zhu et al., “Multipotential differentiation of adipose tissue-derived stem cells,” Keio Journal of Medicine, vol. 54, no. 3, pp. 132–141, 2005.). Once isolated from the fat, ASCs have the capacity to form fat cells, but also bone, cartilage and muscle cells.

From a therapeutic standpoint, ASCs promote the development of new blood vessels (angiogenesis). ASCs are also not recognized by the immune system and they seem to staunch inflammation. According the Dr. Rohrich and is co-authors, “Clinicians and patients have high expectations that ASCs may well be the answer to curing many recalcitrant diseases or to reconstruct anatomical defects.”

Fortunately, there is great interest in ASCs, and this means that the number of studies that examine ASCs or utilize them for experimental treatments have soared. Unfortunately, there is continued concern about the “true clinical potential” of ASCs. In the words of this new article, “For example, there are questions related to isolation and purification of ASCs, their effect on tumor growth, and the enforcement of FDA regulations.”

Rohrich and others conducted a rather in-depth review of all known clinical trials of ASCs. Thus far, most studies have been performed in Europe and Korea, and only three in the United States, to date. This reflects the stringency of FDA regulations.

Most ASC clinical trials to date have been examined the use of ASCs in plastic surgery. In this case, plastic surgeon-researchers have used ASCs for several types of soft tissue augmentation (breast augmentation, especially after implant removal and regeneration of fat in patients with abnormal fat loss or lipodystrophy). Studies exploring the use of ASCs to promote healing of difficult wounds have been reported as well. ASCs have also been used as in so-called soft tissue engineering or tissue regeneration. In these cases, the results have been inconclusive.

Other medical specialties have also made use of ASCs as treatments for other types of medical conditions. For example, ASCs have been studied for used to treat certain blood and immunologic disorders, heart and vascular problems, and fistulas (abnormal connection between an organ, vessel, or intestine and another structure). There are some other studies that have examined the use of ASCs for generating new bone for use in reconstructive surgery. A few studies have reported promising preliminary results in the treatment of diabetes, multiple sclerosis, and spinal cord injury. Perhaps one of the most encouraging results was the complete absence of serious adverse events related to ASCs in any of these studies.

These results are encouraging, but all of these applications are in their infancy. Globally speaking, less than 300 patients have been treated with ASCs, and no standardized protocol exists for the preparation or clinical applications of ASCs. Additionally, there is no consensus as to the number of ASCs required per treatment, or how many treatments are required for the patient to show clinical improvement. Thus Rohrich and his colleagues have taken a “proceed with caution approach.” They conclude that “further basic science experimental studies with standardized protocols and larger randomized controlled trials need to be performed to ensure safety and efficacy of ASCs in accordance with FDA guidelines.”