Kevin Hicok - Academia.edu (original) (raw)
Papers by Kevin Hicok
Journal of Cellular Biochemistry, Sep 15, 1997
Although the differentiation of mature osteoblasts has been well studied, there is still a need f... more Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1␣,25-dihydroxyvitamin D 3 (1,25(OH 2)D 3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10 ؊8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10 ؊8 M 1,25(OH 2)D 3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH 2)D 3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and -glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH 2)D 3 , or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans. (
Journal of Bone and Mineral Research, Feb 1, 1998
Although the differentiation of mature osteoblasts has been well studied, there is still a need f... more Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1␣,25-dihydroxyvitamin D 3 (1,25(OH 2)D 3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10 ؊8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10 ؊8 M 1,25(OH 2)D 3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH 2)D 3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and -glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH 2)D 3 , or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans. (
Journal of Bone and Mineral Research, Sep 1, 1999
Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor ... more Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein‐2 (BMP‐2) on a conditionally immortalized human marrow stromal cell line, hMS(2–6), which is capable of differentiation to either lineage. BMP‐2 did not affect hMS(2–6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8‐fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP‐2–stimulated expression of ALP mRNA and protein. The effects of BMP‐2 on adipocyte differentiation varied: expression of peroxisome proliferator‐activated receptor γ2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP‐2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.
Investigative Ophthalmology & Visual Science, 2007
Stem Cell Therapy for Autoimmune Disease, 2019
Stem Cells and Development
Stem Cell and Regenerative Medicine
As the field of exosome therapeutics expands, several companies are providing products sourced fr... more As the field of exosome therapeutics expands, several companies are providing products sourced from different cells or tissues, often claiming equivalent or superior safety and efficacy profiles with little data to support these claims. A cursory review of scientific literature quickly reveals that not all exosomes are created equal. Exosomes are secreted by most, if not all cells. The composition of exosome content, however, is variable and dependent upon the originating cell type and its environment. Therefore, understanding the primary critical attributes of the cell source will provide the end-user important information to evaluate the quality, safety, and potential efficacy of the exosome product. Currently, bone marrow-derived mesenchymal stem cells (BM-MSCs) and placenta tissuederived cells are primary cell sources for commercial products. The purpose of this paper is to elucidate important considerations of exosome origin before use.
Veterinary Surgery
Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive i... more Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive improvements in veterinary regenerative medicine. Inherent limitations to long-term culture preclude use for storage. Until cell line creation from primary isolates becomes routine, MSC stasis at cryogenic temperatures is required for this purpose. Many protocols and reagents, including cryoprotectants, used for veterinary MSCs are derived from those for human and rodent cells. Dissimilarities in cryopreservation strategies play a role in variable MSC behaviors. Familiarity with contemporary cryopreservation reagents and processes is essential to an appreciation of their impact on MSC survival and post-cryopreservation behavior. In addition to these points, this review includes a brief history and description of current veterinary stem cell regulation.
Annals of Plastic Surgery, 2015
Pluripotential cells in adipose tissue may be important in long-term volume retention and regener... more Pluripotential cells in adipose tissue may be important in long-term volume retention and regenerative effects of fat grafting. Unfortunately, graft harvest with lipoaspiration significantly depletes the population of stromal vascular cells, which includes adipose stem cells. Stromal vascular fraction (SVF) cells may be isolated from excess lipoaspirate at the point of care and used to replenish fat grafts, a technique termed cell-assisted lipotransfer (CAL). Preclinical and clinical evidence supports the rationale of CAL but clinical adoption of the strategy requires evidence of clinical safety. This prospective, level 1 study reports clinical safety of SVF-enhanced fat grafting using a manual, collagenase-based separation process to isolate autogenous progenitor cells from lipoaspirate at the point of care. One hundred sixty-four subjects underwent 174 SVF-enhanced autologous fat grafting procedures at the university stem cell center between August 2009 and November 2014 for a variety of cosmetic and reconstructive indications. Cell-assisted lipotransfer was performed for a variety of cosmetic and reconstructive indications. The mean time of the SVF isolation process was 91 minutes. Because of the frequent concomitant procedures, the average operating room time increased by only 11 minutes. Mean follow-up was 19.9 months. There were no major complications and 6 minor complications. No collagenase or neutral protease related complications were observed. This series of 174 CAL cases demonstrates that SVF cell isolation using a standardized, manual, collagenase-based process at the POC is equivalent in safety compared to nonenhanced fat grafting. These results support expanded use of CAL in the clinical research setting.
2012 38th Annual Northeast Bioengineering Conference (NEBEC), 2012
ABSTRACT Laboratory investigations have been conducted to assess the viability of Adipose Derived... more ABSTRACT Laboratory investigations have been conducted to assess the viability of Adipose Derived stem and Regenerative Cells (ADRCs) and adipocytes harvested using a third generation (VASER®) Ultrasound Assisted Liposuction system. Methods: Lipoaspirate samples were collected from consented patients undergoing elective liposuction procedures. The VASER system was set at 60-70% amplitude in the pulsed mode; the aspiration system was set for a vacuum level of 15 inHg or less. Laboratory analysis included measurement of free lipid volume; lipolysis assay to determine the health (quality) of the aspirated adipose tissue; analysis of ADRCs released from adipose tissue for number and viability; CFU-F assay to evaluate adherent cell population of ADRCs; and flow cytometery and cytological analysis, including cell surface protein examination and H&E staining. Results: The lipolysis assay found the adipocytes to be metabolically active and possessing a mean correlative viability of 85.1±11%; ADRC viability was 87.4±4.5%. Further evaluation demonstrated that ADRCs were composed of different stromal vascular and hematopoietic subpopulations proportionate to cells obtained from syringe acquired tissue. Conclusions: Collectively these data indicate that adipose tissue acquired using VASER methodology is viable at harvest and suitable as a source of Adipose Derived stem and Regenerative Cells (ADRCs) and fat grafting material.
Thesis (M.S.)--University of Minnesota. Includes bibliographical references (leaves 69-83).
Laboratory investigations have been conducted to assess the viability of Adipose Derived stem and... more Laboratory investigations have been conducted to assess the viability of Adipose Derived stem and Regenerative Cells (ADRCs) and adipocytes harvested using a third generation (VASER®) Ultrasound Assisted Liposuction system. Methods: Lipoaspirate samples were collected from consented patients undergoing elective liposuction procedures. The VASER system was set at 60-70% amplitude in the pulsed mode; the aspiration system was set for a vacuum level of 15 inHg or less. Laboratory analysis included measurement of free lipid volume; lipolysis assay to determine the health (quality) of the aspirated adipose tissue; analysis of ADRCs released from adipose tissue for number and viability; CFU-F assay to evaluate adherent cell population of ADRCs; and flow cytometery and cytological analysis, including cell surface protein examination and H&E staining. Results: The lipolysis assay found the adipocytes to be metabolically active and possessing a mean correlative viability of 85.1±11%; ADRC vi...
Techniques in Aesthetic Plastic Surgery Series: Facial Rejuvenation with Fillers, 2009
Adipose-Derived Stem Cells, 2010
The popularity of nonhematopoietic, adult tissue-derived stem and progenitor cells for use as a c... more The popularity of nonhematopoietic, adult tissue-derived stem and progenitor cells for use as a cellular research tool, and ultimately as a clinical therapeutic, has increased exponentially over the past decade. Almost all adult-derived stem/progenitor cells (autologous and allogeneic), with one exception, require at least some ex vivo expansion or further manipulation prior to use to satisfy efficacy and safety requirements for preclinical or clinical use. The principal reason is the relatively low frequency of these therapeutically valuable cells within any given adult tissue, except for adipose tissue, which has been shown to have at least two log greater concentrations of these progenitor cells. Therefore, use of autologous adipose-derived cells as both a research tool and cell therapeutic is feasible and has been shown to be both safe and efficacious in preclinical and clinical models of injury and disease. The development and utilization of automated processes and instrumentation such as Cytori Therapeutics' Celution® System to reduce variability and increase quality of the recovered cells is requisite for clinical use and preferred by basic researchers. Here, use of an automated, closed processing platform for isolation and concentration of adipose-derived stem and regenerative cells is described, including a profile of the isolated cells immediately prior to use, and commonly used methods to quantify and qualitatively assess the recovered cells.
Topics in Bone Biology, 2007
these cells to determine similarity to hES cells generated via sexual reproduction has not yet ad... more these cells to determine similarity to hES cells generated via sexual reproduction has not yet advanced far. Furthermore, teratoma formation by hES cells remains a safety issue, so that large-scale clinical trials involving these cells cannot be undertaken until the safety issue is resolved. Adults also have stem cells. Hematopoietic stem cells in the bone marrow that can reconstitute the immune system have been known and studied for many years [13]. Stem cells in the liver allow rapid regeneration after liver surgery; stem cells in the dermis undergo continuous cell division and differentiation to replace skin cells; and mesenchymal stem cells (MSCs) in bone provide osteoblasts for bone remodeling throughout life. Until the mid-1980s, these stem cells were thought to be committed to regenerating only the tissue in which they resided and were believed to be unable to differentiate toward cell fates not associated with their germinal layer of origin. Their potential as "true" stem cells was therefore not realized. In the 1990s, the molecular mechanisms involved in cellular differentiation began to be understood more fully. Moreover, development of in vitro differentiation assays helped cell biologists and tissue engineers realize the therapeutic potential of these cells. This chapter will review the successes, challenges, and future prospects of using stem cells in the tissue engineering of bone, cartilage, tendon, and ligament.
Stem Cells and Tissue Repair, 2014
Adipose tissue sciences have rapidly expanded since the identification of regenerative cells cont... more Adipose tissue sciences have rapidly expanded since the identification of regenerative cells contained within the stromal vascular fraction (SVF) of fat. Isolation of the SVF, containing adipose-derived stem cells (ADSC), can be accomplished efficiently in the operating room or in the laboratory through enzymatic digestion of the adipose tissue and concentration of SVF. Cells can be directly re-injected as a mesotherapeutic agent, recombined with a tissue scaffold (e.g., cell-enriched fat grafts) or expanded in culture for tissue-engineered cell therapeutics. The potential for cell therapy is under current investigation by researchers around the world. This chapter reviews laboratory methods for isolating ADSCs and the ongoing clinical trials evaluating cell therapeutic efficacy across many specialties, including cardiology, neurology, immunology, tissue engineering, sports medicine, and plastic and reconstructive surgery.
Advances in Wound Care, 2014
Objective: To develop a closed, automated system that standardizes the processing of human adipos... more Objective: To develop a closed, automated system that standardizes the processing of human adipose tissue to obtain and concentrate regenerative cells suitable for clinical treatment of thermal and radioactive burn wounds. Approach: A medical device was designed to automate processing of adipose tissue to obtain a clinical-grade cell output of stromal vascular cells that may be used immediately as a therapy for a number of conditions, including nonhealing wounds resulting from radiation damage. Results: The Celution(®) System reliably and reproducibly generated adipose-derived regenerative cells (ADRCs) from tissue collected manually and from three commercial power-assisted liposuction devices. The entire process of introducing tissue into the system, tissue washing and proteolytic digestion, isolation and concentration of the nonadipocyte nucleated cell fraction, and return to the patient as a wound therapeutic, can be achieved in approximately 1.5 h. An alternative approach that applies ultrasound energy in place of enzymatic digestion demonstrates extremely poor efficiency cell extraction. Innovation: The Celution System is the first medical device validated and approved by multiple international regulatory authorities to generate autologous stromal vascular cells from adipose tissue that can be used in a real-time bedside manner. Conclusion: Initial preclinical and clinical studies using ADRCs obtained using the automated tissue processing Celution device described herein validate a safe and effective manner to obtain a promising novel cell-based treatment for wound healing.
Tissue Engineering, 2004
Adult subcutaneous fat tissue is an abundant source of multipotent cells. Previous studies from o... more Adult subcutaneous fat tissue is an abundant source of multipotent cells. Previous studies from our laboratory have shown that, in vitro, adipose-derived adult stem (ADAS) cells express bone marker proteins including alkaline phosphatase, type I collagen, osteopontin, and osteocalcin and produce a mineralized matrix as shown by alizarin red staining. In the current study, the ADAS cell ability to form osteoid in vivo was determined. ADAS cells were isolated from liposuction waste of three individual donors and expanded in vitro before implantation. Equal numbers of cells (3 x 10(6)) were loaded onto either hydroxyapatite/tricalcium phosphate (HA-TCP) cubes or the collagen/HA-TCP composite matrix, Collagraft, and then implanted subcutaneously into SCID mice. After 6 weeks, implants were removed, fixed, and demineralized and sectioned for hematoxylin and eosin staining. Osteoid formation was observed in 80% of HA-TCP implants loaded with ADAS cells. Only 20% of Collagraft implants were positive for the presence of osteoid matrix. Whereas 100% of HA-TCP implants loaded with hFOB 1.19 cells formed osteoid, Collagraft loaded with hFOB 1.19 cells displayed a high degree of adipose tissue within the matrix. Immunostaining of serial sections for human nuclear antigen demonstrated that the osteoid contained human cells. Osteoid formation was not observed in control HA-TCP or Collagraft matrices implanted without cells. In summary, the data demonstrate the ability of ADAS cells to form osteoid matrix in vivo. Because of their abundance and accessibility, ADAS cells may prove to be a novel cell therapeutic for bone repair and regeneration.
Plastic and Reconstructive Surgery, 2014
Suday, M arch 9, 2014 effect. The angiogenic effect of PRP itself and PRP-treated ASCs will enhan... more Suday, M arch 9, 2014 effect. The angiogenic effect of PRP itself and PRP-treated ASCs will enhance the vascular supply to maintain the adipocyte survival within fat graft. Furthermore, the stemness effect of PRP increases the renewal and differentiation capabilities of ASCs which can be the cell depot required in fat graft survival. 131 adipose derived Stem cell count is influenced by receipt of chemotherapy in Breast cancer Patients
Plastic and Reconstructive Surgery, 2010
ABSTRACT Purpose: Syringe aspiration (SA) is a commonly used method for harvesting adipose tissue... more ABSTRACT Purpose: Syringe aspiration (SA) is a commonly used method for harvesting adipose tissue for autologous fat grafting. Water-jet assisted liposuction (WAL) is becoming more widely used and is considered a more gentle method for removing adipose tissue, though this observation lacks supporting data. Differences in tissue quality between these WAL and SA acquisition methods were determined by assessing the biologic properties of the mature fat cells, and the stromal vascular fraction cells found within or loosely attached to the adipose tissue. Methods: Tissue was collected by both procedures on opposite sides of the same bilateral depot (N=5). Adipose tissue quality was evaluated by the functional lipolysis assay. Tissue from both harvesting methods were processed with the Celution 800/CRS device (Cytori Therapeutics, CA) and the nucleated cell output was evaluated by cell yield, viability, flow cytometry and clonogenic assays. For loosely attached cells and the cells lost in the wetting solution, the number of viable cells was determined. Results: Adipose harvested by both methods had comparable lipolysis activity. In addition, similar numbers of viable adipose-derived stem and regenerative cells (ADRCs) were obtained from intact tissues harvested by both methods. Significantly more viable loose cells were collected from SA harvested samples (27.5310.95 x104, with 91.426.55% viability) compared to WAL harvested tissue (2.261.59x104, with 62.3618.30% viability, both p<0.001). By CFU-F assay the total number of ADRCs from SA samples (1.900.21%) was significantly higher than that of WAL samples (1.120.23%, p<0.001). Conclusion: The health of intact mature adipose tissue obtained by both harvest methods is comparable. Furthermore, the viability, concentration within the intact tissue parcels, and the molecular phenotype of stromal vascular cells obtained from this tissue is also comparable. However, the use of WAL resulted in significantly less loosely adherent cells remaining attached to the adipose tissue after processing. As a result more clonogenic adipose stem and regenerative cells were found in the SA harvested graft. Therefore, while WAL harvested graft tissue appears enriched in regard to mature fat cells; a ramification of this concentration and purification may be the significant loss of ADRCs. Further studies in vivo are warranted.
Journal of Cellular Biochemistry, Sep 15, 1997
Although the differentiation of mature osteoblasts has been well studied, there is still a need f... more Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1␣,25-dihydroxyvitamin D 3 (1,25(OH 2)D 3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10 ؊8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10 ؊8 M 1,25(OH 2)D 3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH 2)D 3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and -glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH 2)D 3 , or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans. (
Journal of Bone and Mineral Research, Feb 1, 1998
Although the differentiation of mature osteoblasts has been well studied, there is still a need f... more Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1␣,25-dihydroxyvitamin D 3 (1,25(OH 2)D 3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10 ؊8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10 ؊8 M 1,25(OH 2)D 3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH 2)D 3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and -glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH 2)D 3 , or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans. (
Journal of Bone and Mineral Research, Sep 1, 1999
Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor ... more Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein‐2 (BMP‐2) on a conditionally immortalized human marrow stromal cell line, hMS(2–6), which is capable of differentiation to either lineage. BMP‐2 did not affect hMS(2–6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8‐fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP‐2–stimulated expression of ALP mRNA and protein. The effects of BMP‐2 on adipocyte differentiation varied: expression of peroxisome proliferator‐activated receptor γ2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP‐2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.
Investigative Ophthalmology & Visual Science, 2007
Stem Cell Therapy for Autoimmune Disease, 2019
Stem Cells and Development
Stem Cell and Regenerative Medicine
As the field of exosome therapeutics expands, several companies are providing products sourced fr... more As the field of exosome therapeutics expands, several companies are providing products sourced from different cells or tissues, often claiming equivalent or superior safety and efficacy profiles with little data to support these claims. A cursory review of scientific literature quickly reveals that not all exosomes are created equal. Exosomes are secreted by most, if not all cells. The composition of exosome content, however, is variable and dependent upon the originating cell type and its environment. Therefore, understanding the primary critical attributes of the cell source will provide the end-user important information to evaluate the quality, safety, and potential efficacy of the exosome product. Currently, bone marrow-derived mesenchymal stem cells (BM-MSCs) and placenta tissuederived cells are primary cell sources for commercial products. The purpose of this paper is to elucidate important considerations of exosome origin before use.
Veterinary Surgery
Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive i... more Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive improvements in veterinary regenerative medicine. Inherent limitations to long-term culture preclude use for storage. Until cell line creation from primary isolates becomes routine, MSC stasis at cryogenic temperatures is required for this purpose. Many protocols and reagents, including cryoprotectants, used for veterinary MSCs are derived from those for human and rodent cells. Dissimilarities in cryopreservation strategies play a role in variable MSC behaviors. Familiarity with contemporary cryopreservation reagents and processes is essential to an appreciation of their impact on MSC survival and post-cryopreservation behavior. In addition to these points, this review includes a brief history and description of current veterinary stem cell regulation.
Annals of Plastic Surgery, 2015
Pluripotential cells in adipose tissue may be important in long-term volume retention and regener... more Pluripotential cells in adipose tissue may be important in long-term volume retention and regenerative effects of fat grafting. Unfortunately, graft harvest with lipoaspiration significantly depletes the population of stromal vascular cells, which includes adipose stem cells. Stromal vascular fraction (SVF) cells may be isolated from excess lipoaspirate at the point of care and used to replenish fat grafts, a technique termed cell-assisted lipotransfer (CAL). Preclinical and clinical evidence supports the rationale of CAL but clinical adoption of the strategy requires evidence of clinical safety. This prospective, level 1 study reports clinical safety of SVF-enhanced fat grafting using a manual, collagenase-based separation process to isolate autogenous progenitor cells from lipoaspirate at the point of care. One hundred sixty-four subjects underwent 174 SVF-enhanced autologous fat grafting procedures at the university stem cell center between August 2009 and November 2014 for a variety of cosmetic and reconstructive indications. Cell-assisted lipotransfer was performed for a variety of cosmetic and reconstructive indications. The mean time of the SVF isolation process was 91 minutes. Because of the frequent concomitant procedures, the average operating room time increased by only 11 minutes. Mean follow-up was 19.9 months. There were no major complications and 6 minor complications. No collagenase or neutral protease related complications were observed. This series of 174 CAL cases demonstrates that SVF cell isolation using a standardized, manual, collagenase-based process at the POC is equivalent in safety compared to nonenhanced fat grafting. These results support expanded use of CAL in the clinical research setting.
2012 38th Annual Northeast Bioengineering Conference (NEBEC), 2012
ABSTRACT Laboratory investigations have been conducted to assess the viability of Adipose Derived... more ABSTRACT Laboratory investigations have been conducted to assess the viability of Adipose Derived stem and Regenerative Cells (ADRCs) and adipocytes harvested using a third generation (VASER®) Ultrasound Assisted Liposuction system. Methods: Lipoaspirate samples were collected from consented patients undergoing elective liposuction procedures. The VASER system was set at 60-70% amplitude in the pulsed mode; the aspiration system was set for a vacuum level of 15 inHg or less. Laboratory analysis included measurement of free lipid volume; lipolysis assay to determine the health (quality) of the aspirated adipose tissue; analysis of ADRCs released from adipose tissue for number and viability; CFU-F assay to evaluate adherent cell population of ADRCs; and flow cytometery and cytological analysis, including cell surface protein examination and H&E staining. Results: The lipolysis assay found the adipocytes to be metabolically active and possessing a mean correlative viability of 85.1±11%; ADRC viability was 87.4±4.5%. Further evaluation demonstrated that ADRCs were composed of different stromal vascular and hematopoietic subpopulations proportionate to cells obtained from syringe acquired tissue. Conclusions: Collectively these data indicate that adipose tissue acquired using VASER methodology is viable at harvest and suitable as a source of Adipose Derived stem and Regenerative Cells (ADRCs) and fat grafting material.
Thesis (M.S.)--University of Minnesota. Includes bibliographical references (leaves 69-83).
Laboratory investigations have been conducted to assess the viability of Adipose Derived stem and... more Laboratory investigations have been conducted to assess the viability of Adipose Derived stem and Regenerative Cells (ADRCs) and adipocytes harvested using a third generation (VASER®) Ultrasound Assisted Liposuction system. Methods: Lipoaspirate samples were collected from consented patients undergoing elective liposuction procedures. The VASER system was set at 60-70% amplitude in the pulsed mode; the aspiration system was set for a vacuum level of 15 inHg or less. Laboratory analysis included measurement of free lipid volume; lipolysis assay to determine the health (quality) of the aspirated adipose tissue; analysis of ADRCs released from adipose tissue for number and viability; CFU-F assay to evaluate adherent cell population of ADRCs; and flow cytometery and cytological analysis, including cell surface protein examination and H&E staining. Results: The lipolysis assay found the adipocytes to be metabolically active and possessing a mean correlative viability of 85.1±11%; ADRC vi...
Techniques in Aesthetic Plastic Surgery Series: Facial Rejuvenation with Fillers, 2009
Adipose-Derived Stem Cells, 2010
The popularity of nonhematopoietic, adult tissue-derived stem and progenitor cells for use as a c... more The popularity of nonhematopoietic, adult tissue-derived stem and progenitor cells for use as a cellular research tool, and ultimately as a clinical therapeutic, has increased exponentially over the past decade. Almost all adult-derived stem/progenitor cells (autologous and allogeneic), with one exception, require at least some ex vivo expansion or further manipulation prior to use to satisfy efficacy and safety requirements for preclinical or clinical use. The principal reason is the relatively low frequency of these therapeutically valuable cells within any given adult tissue, except for adipose tissue, which has been shown to have at least two log greater concentrations of these progenitor cells. Therefore, use of autologous adipose-derived cells as both a research tool and cell therapeutic is feasible and has been shown to be both safe and efficacious in preclinical and clinical models of injury and disease. The development and utilization of automated processes and instrumentation such as Cytori Therapeutics&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; Celution® System to reduce variability and increase quality of the recovered cells is requisite for clinical use and preferred by basic researchers. Here, use of an automated, closed processing platform for isolation and concentration of adipose-derived stem and regenerative cells is described, including a profile of the isolated cells immediately prior to use, and commonly used methods to quantify and qualitatively assess the recovered cells.
Topics in Bone Biology, 2007
these cells to determine similarity to hES cells generated via sexual reproduction has not yet ad... more these cells to determine similarity to hES cells generated via sexual reproduction has not yet advanced far. Furthermore, teratoma formation by hES cells remains a safety issue, so that large-scale clinical trials involving these cells cannot be undertaken until the safety issue is resolved. Adults also have stem cells. Hematopoietic stem cells in the bone marrow that can reconstitute the immune system have been known and studied for many years [13]. Stem cells in the liver allow rapid regeneration after liver surgery; stem cells in the dermis undergo continuous cell division and differentiation to replace skin cells; and mesenchymal stem cells (MSCs) in bone provide osteoblasts for bone remodeling throughout life. Until the mid-1980s, these stem cells were thought to be committed to regenerating only the tissue in which they resided and were believed to be unable to differentiate toward cell fates not associated with their germinal layer of origin. Their potential as "true" stem cells was therefore not realized. In the 1990s, the molecular mechanisms involved in cellular differentiation began to be understood more fully. Moreover, development of in vitro differentiation assays helped cell biologists and tissue engineers realize the therapeutic potential of these cells. This chapter will review the successes, challenges, and future prospects of using stem cells in the tissue engineering of bone, cartilage, tendon, and ligament.
Stem Cells and Tissue Repair, 2014
Adipose tissue sciences have rapidly expanded since the identification of regenerative cells cont... more Adipose tissue sciences have rapidly expanded since the identification of regenerative cells contained within the stromal vascular fraction (SVF) of fat. Isolation of the SVF, containing adipose-derived stem cells (ADSC), can be accomplished efficiently in the operating room or in the laboratory through enzymatic digestion of the adipose tissue and concentration of SVF. Cells can be directly re-injected as a mesotherapeutic agent, recombined with a tissue scaffold (e.g., cell-enriched fat grafts) or expanded in culture for tissue-engineered cell therapeutics. The potential for cell therapy is under current investigation by researchers around the world. This chapter reviews laboratory methods for isolating ADSCs and the ongoing clinical trials evaluating cell therapeutic efficacy across many specialties, including cardiology, neurology, immunology, tissue engineering, sports medicine, and plastic and reconstructive surgery.
Advances in Wound Care, 2014
Objective: To develop a closed, automated system that standardizes the processing of human adipos... more Objective: To develop a closed, automated system that standardizes the processing of human adipose tissue to obtain and concentrate regenerative cells suitable for clinical treatment of thermal and radioactive burn wounds. Approach: A medical device was designed to automate processing of adipose tissue to obtain a clinical-grade cell output of stromal vascular cells that may be used immediately as a therapy for a number of conditions, including nonhealing wounds resulting from radiation damage. Results: The Celution(®) System reliably and reproducibly generated adipose-derived regenerative cells (ADRCs) from tissue collected manually and from three commercial power-assisted liposuction devices. The entire process of introducing tissue into the system, tissue washing and proteolytic digestion, isolation and concentration of the nonadipocyte nucleated cell fraction, and return to the patient as a wound therapeutic, can be achieved in approximately 1.5 h. An alternative approach that applies ultrasound energy in place of enzymatic digestion demonstrates extremely poor efficiency cell extraction. Innovation: The Celution System is the first medical device validated and approved by multiple international regulatory authorities to generate autologous stromal vascular cells from adipose tissue that can be used in a real-time bedside manner. Conclusion: Initial preclinical and clinical studies using ADRCs obtained using the automated tissue processing Celution device described herein validate a safe and effective manner to obtain a promising novel cell-based treatment for wound healing.
Tissue Engineering, 2004
Adult subcutaneous fat tissue is an abundant source of multipotent cells. Previous studies from o... more Adult subcutaneous fat tissue is an abundant source of multipotent cells. Previous studies from our laboratory have shown that, in vitro, adipose-derived adult stem (ADAS) cells express bone marker proteins including alkaline phosphatase, type I collagen, osteopontin, and osteocalcin and produce a mineralized matrix as shown by alizarin red staining. In the current study, the ADAS cell ability to form osteoid in vivo was determined. ADAS cells were isolated from liposuction waste of three individual donors and expanded in vitro before implantation. Equal numbers of cells (3 x 10(6)) were loaded onto either hydroxyapatite/tricalcium phosphate (HA-TCP) cubes or the collagen/HA-TCP composite matrix, Collagraft, and then implanted subcutaneously into SCID mice. After 6 weeks, implants were removed, fixed, and demineralized and sectioned for hematoxylin and eosin staining. Osteoid formation was observed in 80% of HA-TCP implants loaded with ADAS cells. Only 20% of Collagraft implants were positive for the presence of osteoid matrix. Whereas 100% of HA-TCP implants loaded with hFOB 1.19 cells formed osteoid, Collagraft loaded with hFOB 1.19 cells displayed a high degree of adipose tissue within the matrix. Immunostaining of serial sections for human nuclear antigen demonstrated that the osteoid contained human cells. Osteoid formation was not observed in control HA-TCP or Collagraft matrices implanted without cells. In summary, the data demonstrate the ability of ADAS cells to form osteoid matrix in vivo. Because of their abundance and accessibility, ADAS cells may prove to be a novel cell therapeutic for bone repair and regeneration.
Plastic and Reconstructive Surgery, 2014
Suday, M arch 9, 2014 effect. The angiogenic effect of PRP itself and PRP-treated ASCs will enhan... more Suday, M arch 9, 2014 effect. The angiogenic effect of PRP itself and PRP-treated ASCs will enhance the vascular supply to maintain the adipocyte survival within fat graft. Furthermore, the stemness effect of PRP increases the renewal and differentiation capabilities of ASCs which can be the cell depot required in fat graft survival. 131 adipose derived Stem cell count is influenced by receipt of chemotherapy in Breast cancer Patients
Plastic and Reconstructive Surgery, 2010
ABSTRACT Purpose: Syringe aspiration (SA) is a commonly used method for harvesting adipose tissue... more ABSTRACT Purpose: Syringe aspiration (SA) is a commonly used method for harvesting adipose tissue for autologous fat grafting. Water-jet assisted liposuction (WAL) is becoming more widely used and is considered a more gentle method for removing adipose tissue, though this observation lacks supporting data. Differences in tissue quality between these WAL and SA acquisition methods were determined by assessing the biologic properties of the mature fat cells, and the stromal vascular fraction cells found within or loosely attached to the adipose tissue. Methods: Tissue was collected by both procedures on opposite sides of the same bilateral depot (N=5). Adipose tissue quality was evaluated by the functional lipolysis assay. Tissue from both harvesting methods were processed with the Celution 800/CRS device (Cytori Therapeutics, CA) and the nucleated cell output was evaluated by cell yield, viability, flow cytometry and clonogenic assays. For loosely attached cells and the cells lost in the wetting solution, the number of viable cells was determined. Results: Adipose harvested by both methods had comparable lipolysis activity. In addition, similar numbers of viable adipose-derived stem and regenerative cells (ADRCs) were obtained from intact tissues harvested by both methods. Significantly more viable loose cells were collected from SA harvested samples (27.5310.95 x104, with 91.426.55% viability) compared to WAL harvested tissue (2.261.59x104, with 62.3618.30% viability, both p<0.001). By CFU-F assay the total number of ADRCs from SA samples (1.900.21%) was significantly higher than that of WAL samples (1.120.23%, p<0.001). Conclusion: The health of intact mature adipose tissue obtained by both harvest methods is comparable. Furthermore, the viability, concentration within the intact tissue parcels, and the molecular phenotype of stromal vascular cells obtained from this tissue is also comparable. However, the use of WAL resulted in significantly less loosely adherent cells remaining attached to the adipose tissue after processing. As a result more clonogenic adipose stem and regenerative cells were found in the SA harvested graft. Therefore, while WAL harvested graft tissue appears enriched in regard to mature fat cells; a ramification of this concentration and purification may be the significant loss of ADRCs. Further studies in vivo are warranted.