David T. Harris

Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation

BackgroundHuman adipose tissue is an ideal autologous source of mesenchymal stem cells (MSCs) for various regenerative medicine and tissue engineering strategies. Aged patients are one of the primary target populations for many promising applications. It has long been known that advanced age is negatively correlated with an organism’s reparative and regenerative potential, but little and conflicting information is available about the effects of age on the quality of human adipose tissue derived MSCs (hAT-MSCs).MethodsTo study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs from young (<30xa0years), adult (35-50xa0years) and aged (>60xa0years) individuals were investigated. MSCs were characterized for expression of the genes p16INK4a and p21 along with measurements of population doublings (PD), superoxide dismutase (SOD) activity, cellular senescence and differentiation potential.ResultsAged MSCs displayed senescent features when compared with cells isolated from young donors, concomitant with reduced viability and proliferation. These features were also associated with significantly reduced differentiation potential in aged MSCs compared to young MSCs.ConclusionsIn conclusion, advancing age negatively impacts stem cell function and such age related alterations may be detrimental for successful stem cell therapies.

Umbilical cord blood: a unique source of pluripotent stem cells for regenerative medicine.

It is estimated that almost 1 in 3 individuals in the United States might benefit from regenerative medicine therapy. Unfortunately, embryonic stem (ES) cell therapies are currently limited by ethical, political, biological and regulatory hurdles. Thus, for the foreseeable future, the march of regenerative medicine to the clinic will depend upon the development of non-ES cell therapies. Current sources of non-ES cells easily available in large numbers can be found in the bone marrow, adipose tissue and umbilical cord blood. Each of these types of stem cells has already begun to be utilized to treat a variety of diseases. This review will show that cord blood (CB) contains multiple populations of ES-like and other pluripotential stem cells, capable of giving rise to hematopoietic, epithelial, endothelial, and neural tissues both in vitro and in vivo. Cumulatively, the identification and isolation of these populations of pluripotent stem cells within cord blood represents a scientific breakthrough that could potentially impact every field of medicine, via their use in regenerative medicine. Thus, CB stem cells are amenable to treatment of a wide variety of diseases including cardiovascular, hepatic, ophthalmic, orthopaedic, neurological and endocrine diseases.

Comparison of human mesenchymal stem cells derived from adipose and cord tissue.

BACKGROUND AIMSnStem cell therapies can provide an alternative approach for repair and regeneration of tissues and organs. Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies. Although bone marrow-derived MSCs have multi-lineage differentiation potential, bone marrow is not an optimal source because of the isolation process and low yield. The goal of this study was to investigate comparatively for the first time the in vitro regenerative potential of human MSCs from two other sources: umbilical cord tissue and adipose tissue.nnnMETHODSnCells from each tissue were isolated with 100% efficiency and characterized by fluorescence activated cell sorting (FACS) analysis for CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105. Growth characteristics were investigated by population doublings, saturation density and plating efficiency. MSCs derived from both types of tissues were assessed for differentiation potential qualitatively and quantitatively.nnnRESULTSnFACS analysis showed no differences in expression of CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105 between cord tissue MSCs (CT-MSCs) and adipose tissue MSCs (AT-MSCs). CT-MSCs showed more proliferative potential than AT-MSCs. When cultured in low numbers to determine colony-forming units (CFUs), CT-MSCs showed less CFUs than AT-MSCs. Cells from both sources efficiently differentiated into adipose, bone, cartilage and neuronal structures as determined with histochemistry, immunofluorescence and real-time reverse transcriptase polymerase chain reaction.nnnCONCLUSIONSnMSCs can easily be obtained from umbilical cord and adipose tissues, and it appears that both tissues are suitable sources of stem cells for potential use in regenerative medicine.

Cryopreservation of whole adipose tissue for future use in regenerative medicine

BACKGROUNDnHuman adipose tissue (AT) is an ideal stem cell source for autologous cell-based therapies. The preferred setting for tissue engineering and regenerative medicine applications is the availability of clinically acceptable off-the-shelf cells and cell products. As AT is not always available for use, cryopreserved tissue represents an alternative approach. The aim of the present study was to compare the different properties of mesenchymal stem cells (MSCs) isolated from cryopreserved AT. We have measured cell recovery, viability, phenotype, proliferative potential, and differentiation into mesenchymal (adipogenic, osteogenic, chondrogenic) and nonmesenchymal (neuron-like cells) lineages.nnnMATERIALS AND METHODSnAT (n = 10) was harvested from donors and either processed fresh or cryopreserved in liquid nitrogen dewars. Both fresh and thawed tissues were enzymatically digested. MSCs were analyzed by fluorescence-activated cell sorting for CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90, and CD105 expression. Growth characteristics of both groups were investigated for population doublings, doubling time, saturation density, and plating efficiency. MSCs derived from fresh and thawed tissues were assessed for differentiation potential both qualitatively and quantitatively.nnnRESULTSnAdherent cells from fresh and thawed tissues displayed similar fibroblastic morphology. Cryopreservation did not alter expression of phenotypic markers. Similarly, the proliferative potential of MSCs was not compromised by cryopreservation. Furthermore, cryopreservation did not alter the differentiation capability of MSCs as determined with histochemistry, immunofluorescence, and real time reverse transcriptase-polymerase chain reaction.nnnCONCLUSIONSnWe conclude that human AT could be successfully cryopreserved for future clinical application and the recovered MSCs were equivalent in functionality to the freshly processed MSCs.

Utility of cryopreserved umbilical cord tissue for regenerative medicine.

MSCs can be isolated from adult sources such as bone marrow and adipose tissue. In contrast to these adult tissue sources, harvesting MSCs from cord tissue is a non-invasive procedure and poses no risk to the donor. Stem cell banks offer the opportunity to cryopreserve cord tissue as a source of MSCs for future autologous or allogeneic stem cell based regenerative medicine applications. There is little published data however, characterizing MSCs isolated from cryopreserved cord tissue. The goal of this study was to determine if MSCs isolated from cryopreserved cord tissue are functionally equivalent to MSCs isolated from fresh cord tissue. Umbilical cords were collected from 10 donors. Cords were segmented into 4-6 inch pieces and either cryopreserved or used immediately. Fresh and thawed cord segments were cultured in 7-14 days for outgrowth of MSCs. MSCs were analyzed by FACS for CD45, CD73, CD90 and CD105 expression. FACs analysis confirmed cells isolated from both fresh and frozen tissue expressed MSC markers. Adherent cells were obtained from both fresh and cryopreserved cord tissue segments at a similar plating efficiency. There was no difference in either the number or time of population doublings. MSCs isolated from fresh and frozen tissue were capable of differentiating along adipogenic, chondrogenic, osteogenic and neurogenic pathways, as confirmed by histology and RT-PCR analysis of tissue specific mRNAs. No significant functional differences were observed between MSCs from frozen cord tissue as compared to fresh cord tissue. Cryopreserving cord tissue allows for isolation of MSCs at the point of care when the specific clinical application is known. This may be advantageous as MSC isolation protocols continue to be optimized dependent on intended use.

Umbilical Cord Tissue Mesenchymal Stem Cells: Characterization and Clinical Applications

Umbilical cord tissue (CT) can provide a virtually unlimited source of multipotent mesenchymal stem cells (MSC) that can potentially be used in a variety of regenerative medicine and tissue engineering applications. Cord tissue segments can be frozen and preserved in liquid nitrogen dewars for prolonged periods of time, having been frozen in time at the peak of biological activity. CT stem cells are capable of giving rise to various mesenchymal and non-mesenchymal cell lineages including bone, cartilage, fat and neurons. Thus, CT stem cells are candidates to develop stem cell-based therapies for a wide variety of diseases including cardiovascular, ophthalmic, orthopedic and neurological applications. CT is currently being used in several regenerative medicine clinical studies, examples of which include treatment of graftversus- host disease and non-healing bone fractures. CT represents an additional source of stem cells that have both immediate and future applications for the individual donor.

Subcutaneous Adipose Tissue–Derived Stem Cell Utility Is Independent of Anatomical Harvest Site

Abstract One of the challenges for tissue engineering and regenerative medicine is to obtain suitably large cell numbers for therapy. Mesenchymal stem cells (MSCs) can easily be expanded in vitro to obtain large numbers of cells, but this approach may induce cellular senescence. The characteristics of cells are dependent on variables like age, body mass index (BMI), and disease conditions, however, and in the case of adipose tissue–derived stem cells (ASCs), anatomical harvest site is also an important variable that can affect the regenerative potential of isolated cells. We therefore had kept the parameters (age, BMI, disease conditions) constant in this study to specifically assess influence of anatomical sites of individual donors on utility of ASCs. Adipose tissue was obtained from multiple anatomical sites in individual donors, and viability and nucleated cell yield were determined. MSC frequency was enumerated using colony forming unit assay and cells were characterized by flow cytometry. Growth characteristics were determined by long-term population doubling analysis of each sample. Finally, MSCs were induced to undergo adipogenic, osteogenic, and chondrogenic differentiation. To validate the findings, these results were compared with similar single harvest sites from multiple individual patients. The results of the current study indicated that MSCs obtained from multiple harvest sites in a single donor have similar morphology and phenotype. All adipose depots in a single donor exhibited similar MSC yield, viability, frequency, and growth characteristics. Equivalent differentiation capacity into osteocytes, adipocytes, and chondrocytes was also observed. On the basis of results, we conclude that it is acceptable to combine MSCs obtained from various anatomical locations in a single donor to obtain suitably large cell numbers required for therapy, avoiding in vitro senescence and lengthy and expensive in vitro culturing and expansion steps.

Fusion of HepG2 cells with mesenchymal stem cells increases cancer-associated and malignant properties: An in vivo metastasis model

In the present study, we have tested the hypothesis that fusion between an altered cell and a mesenchymal stem cell produces a hybrid cell with enhanced characteristics associated with metastatic cancer cells, and we have developed a flexible model for investigating the mechanisms of metastasis. Human HepG2 cells with low metastatic potential were induced to fuse with rat bone marrow mesenchymal stem cells, and the progeny were compared with the parental cells for possession of enhanced in vitro and in vivo characteristics of malignant cells. Compared to the parental cells, the fused cells exhibited enhanced expression of E-cadherin, vimentin, Twist, Snail, matrix metalloproteinase 2 and 9 activities, aneuploidy and enhanced in vitro invasion and migration. In an in vivo xenograft assay, the fused cells generated increased numbers of metastatic liver and lung lesions. This model system is a flexible tool for investigation of the mechanisms of stem cell fusion in carcinogenesis and metastasis and for the discovery of new therapeutic targets to inhibit metastasis.

Effect of mild heat stress on the proliferative and differentiative ability of human mesenchymal stromal cells

BACKGROUND AIMSnMesenchymal stromal cells (MSCs) are an attractive candidate for autologous cell therapy, but regenerative potential can be compromised with extensive in vitro cell passaging. Development of viable cell therapies must address the effect of in vitro passaging to maintain overall functionality of expanded MSCs.nnnMETHODSnWe examined the effect of repeated mild heat shock on the proliferation and differentiation capability of human adipose-derived MSCs. Adipose tissue MSCs were characterized by means of fluorescence activated cell sorting analysis for expression of CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105. Similarly, the expression of SIRT-1, p16(INK4a) and p21 was determined by means of polymerase chain reaction. Measurements of population doubling, doubling time and superoxide dismutase activity were also determined. Differentiation of expanded MSCs into bone and adipose were analyzed qualitatively and quantitatively.nnnRESULTSnThe strategy led to an increase in expression of SIRT-1 concomitant with enhanced viability, proliferation and delayed senescence. The stressed MSCs showed better differentiation into osteoblasts and adipocytes.nnnCONCLUSIONSnThe results indicate that mild heat shock could be used to maintain MSC proliferative and differentiation potential.

Long term human reconstitution and immune aging in NOD-Rag (-)-γ chain (-) mice.

Aging of the human immune system is characterized by a gradual loss of immune function and a skewing of hematopoiesis toward the myeloid lineage, a reduction in the lymphocytic lineage, and progressive increases in senescent memory T cells at the expense of naïve T cells. Both the innate and the adaptive branches of the immune system are affected, including neutrophils, macrophages, dendritic cells and lymphocytes. Mice, the most common research model, although inexpensive, do not necessarily reflect the human immune system in terms of its interaction with infectious agents of human origin or environmental factors. This study analyzed whether a human immune system contained within the NOD-Rag (-)-γ chain (-) mouse model could realistically be used to evaluate the development and therapy of aging-related diseases. To that end lightly irradiated NOD-Rag (-)-γ chain (-) mice were injected intra-hepatically on day 1 of life with purified cord blood-derived CD34(+) stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analyzed quarterly for age-related changes in the hematopoietic and immune systems, and followed for periods up to 18-24 months post-transplant. Flow cytometric analyses were performed for hematopoietic and immune reconstitution. It was observed that NOD-Rag (-)-γ chain (-) mice could be humanized long-term using cord blood stem cells, and that some evidence of immune aging occurred during the life of the mice.