Rejuvenation of young blood on aging organs: Effects, circulating factors, and mechanisms.

Aging causes degenerative changes in organs, leading to a decline in physical function. Over the past two decades, researchers have made significant progress in understanding the rejuvenating effects of young blood on aging organs, benefiting from heterochronic parabiosis models that connect the blood circulation of aged and young rodents. It has been discovered that young blood can partially rejuvenate organs in old animals by regulating important aging-related signaling pathways. Clinical trials have also shown the effectiveness of young blood in treating aging-related diseases. However, the limited availability of young blood poses a challenge to implementing anti-aging therapies on a large scale for older individuals. As a promising alternative, scientists have identified some specific anti-aging circulating factors in young blood that have been shown to promote organ regeneration, reduce inflammation, and alleviate fibrosis associated with aging in animal experiments. While previous reviews have focused primarily on the effects and mechanisms of circulating factors on aging, it is important to acknowledge that studying the rejuvenating effects and mechanisms of young blood has been a significant source of inspiration in this field, and it will continue to be in the future. In recent years, new findings have emerged, further expanding our knowledge in this area. This review aims to summarize the rejuvenating effects and mechanisms of young blood and circulating factors, discussing their similarities and connections, addressing discrepancies in previous studies, outlining future research directions, and highlighting the potential for clinical translation in anti-aging interventions.

Blood-based therapies to combat neurodegenerative diseases.

Neurodegeneration, known as the progressive loss of neurons in terms of their structure and function, is the principal pathophysiological change found in the majority of brain-related disorders. Ageing has been considered the most well-established risk factor in most common neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD). There is currently no effective treatment or cure for these diseases; the approved therapeutic options to date are only for palliative care. Ageing and neurodegenerative diseases are closely intertwined; reversing the aspects of brain ageing could theoretically mitigate age-related neurodegeneration. Ever since the regenerative properties of young blood on aged tissues came to light, substantial efforts have been focused on identifying and characterizing the circulating factors in the young and old systemic milieu that may attenuate or accentuate brain ageing and neurodegeneration. Later studies discovered the superiority of old plasma dilution in tissue rejuvenation, which is achieved through a molecular reset of the systemic proteome. These findings supported the use of therapeutic blood exchange for the treatment of degenerative diseases in older individuals. The first objective of this article is to explore the rejuvenating properties of blood-based therapies in the ageing brains and their therapeutic effects on AD. Then, we also look into the clinical applications, various limitations, and challenges associated with blood-based therapies for AD patients.

Hiding in plain sight: Do recruited dendritic cells surround amyloid plaques in Alzheimer's disease?

Over the past decade, human genome-wide association and expression studies have strongly implicated dysregulation of the innate immune system in the pathogenesis of Alzheimer's disease (AD). Single cell mRNA sequencing studies have identified innate immune cell subtypes that are minimally present in normal healthy brain, but whose numbers greatly increase in association with AD pathology. These AD pathology-associated immune cells are putatively the locus for the immune-related AD risk. While the prevailing view is that these immune cells arise from transformation of resident brain microglia, studies across several decades and using multiple techniques and strategies suggest instead that the pathology-associated immune cells are bone-marrow derived hematopoietic cells that are recruited into brain. We critically review this translational literature, emphasizing the strengths and limitations of techniques used to address recruitment and the experimental designs employed. We conclude that the aggregate evidence points toward recruitment into brain of innate immune cells of the myeloid dendritic cell lineage. Recruitment of dendritic cells and their role in AD pathogenesis has broad implications for our understanding of the etiology and pathobiology of AD that impact the strategies to develop new, immune system-targeted therapeutics for this devastating disease.

Rejuvenating aged stem cells: therapeutic strategies to extend health and lifespan.

Aging is associated with a global decline in stem cell function. To date, several strategies have been proposed to rejuvenate aged stem cells: most of these result in functional improvement of the tissue where the stem cells reside, but the impact on the lifespan of the whole organism has been less clearly established. Here, we review some of the most recent work dealing with interventions that improve the regenerative capacity of aged somatic stem cells in mammals and that might have important translational possibilities. Overall, we underscore that somatic stem cell rejuvenation represents a strategy to improve tissue homeostasis upon aging and present some recent approaches with the potential to affect health span and lifespan of the whole organism.

Epigenetic aging of human blood cells is influenced by the age of the host body.

Allogenic hematopoietic stem cell transplantation is a therapeutic procedure performed over a wide range of donor and recipient age combinations, representing natural experiments of how the age of the recipient affects aging in transplanted donor cells in vivo. We measured DNA methylation and epigenetic aging in donors and recipients and found that biological epigenetic clocks are accelerated in cells transplanted into an older body and decelerated in a younger body. This is the first evidence that the age of the circulating environment influences human epigenetic aging in vivo.

Antiaging Strategies and Remedies: A Landscape of Research Progress and Promise.

Aging is typified by a gradual loss of physiological fitness and accumulation of cellular damage, leading to deteriorated functions and enhanced vulnerability to diseases. Antiaging research has a long history throughout civilization, with many efforts put forth to understand and prevent the effects of aging. Multiple strategies aiming to promote healthy aging and extend the lifespan have been developed including lifestyle adjustments, medical treatments, and social programs. A multitude of antiaging medicines and remedies have also been explored. Here, we use data from the CAS Content Collection to analyze the publication landscape of recent research related to antiaging strategies and treatments. We review the recent advances and delineate trends in research headway of antiaging knowledge and practice across time, geography, and development pipelines. We further assess the state-of-the-art antiaging approaches and explore their correlations with age-related diseases. The landscape of antiaging drugs has been outlined and explored. Well-recognized and novel, currently evaluated antiaging agents have also been summarized. Finally, we review clinical applications of antiaging products with their development pipelines. The objective of this review is to summarize current knowledge on preventive strategies and treatment remedies in the field of aging, to outline challenges and evaluate growth opportunities, in order to further efforts to solve the problems that remain.

SEL1L-HRD1 ER-associated degradation regulates leptin receptor maturation and signaling in POMC neurons in diet-induced obesity.

Endoplasmic reticulum (ER) homeostasis in the hypothalamus has been implicated in the pathogenesis of certain patho-physiological conditions such as diet-induced obesity (DIO) and type 2 diabetes; however, the significance of ER quality control mechanism(s) and its underlying mechanism remain largely unclear and highly controversial in some cases. Moreover, how the biogenesis of nascent leptin receptor in the ER is regulated remains largely unexplored. Here we report that the SEL1L-HRD1 protein complex of the highly conserved ER-associated protein degradation (ERAD) machinery in POMC neurons is indispensable for leptin signaling in diet-induced obesity. SEL1L-HRD1 ERAD is constitutively expressed in hypothalamic POMC neurons. Loss of SEL1L in POMC neurons attenuates leptin signaling and predisposes mice to HFD-associated pathologies including leptin resistance. Mechanistically, newly synthesized leptin receptors, both wildtype and disease-associated human mutant Cys604Ser (Cys602Ser in mice), are misfolding prone and bona fide substrates of SEL1L-HRD1 ERAD. Indeed, defects in SEL1L-HRD1 ERAD markedly impair the maturation of these receptors and causes their ER retention. This study not only uncovers a new role of SEL1L-HRD1 ERAD in the pathogenesis of diet-induced obesity and central leptin resistance, but a new regulatory mechanism for leptin signaling.

Parabiosis Discriminates the Circulating, Endothelial, and Parenchymal Contributions of Endogenous Tissue-Type Plasminogen Activator to Stroke.

BACKGROUND: Intravenous injection of alteplase, a recombinant tPA (tissue-type plasminogen activator) as a thrombolytic agent has revolutionized ischemic stroke management. However, tPA is a more complex enzyme than expected, being for instance able to promote thrombolysis, but at the same time, also able to influence neuronal survival and to affect the integrity of the blood-brain barrier. Accordingly, the respective impact of endogenous tPA expressed/present in the brain parenchyma versus in the circulation during stroke remains debated. METHODS: To address this issue, we used mice with constitutive deletion of tPA (tPANull [tPA-deficient mice]) or conditional deletion of endothelial tPA (VECad [vascular endothelial-Cadherin-Cre-recombinase]-Cre∆tPA). We also developed parabioses between tPANull and wild-type mice (tPAWT), anticipating that a tPAWT donor would restore levels of tPA to normal ones, in the circulation but not in the brain parenchyma of a tPANull recipient. Stroke outcomes were investigated by magnetic resonance imaging in a thrombo-embolic or a thrombotic stroke model, induced by local thrombin injection or FeCl3 application on the endothelium, respectively. RESULTS: First, our data show that endothelial tPA, released into the circulation after stroke onset, plays an overall beneficial role following thrombo-embolic stroke. Accordingly, after 24 hours, tPANull/tPANull parabionts displayed less spontaneous recanalization and reperfusion and larger infarcts compared with tPAWT/tPAWT littermates. However, when associated to tPAWT littermates, tPANull mice had similar perfusion deficits, but less severe brain infarcts. In the thrombotic stroke model, homo- and hetero-typic parabionts did not differ in the extent of brain damages and did not differentially recanalize and reperfuse. CONCLUSIONS: Together, our data reveal that during thromboembolic stroke, endogenous circulating tPA from endothelial cells sustains a spontaneous recanalization and reperfusion of the tissue, thus, limiting the extension of ischemic lesions. In this context, the impact of endogenous parenchymal tPA is limited.

Rejuvenation of cerebromicrovascular function in aged mice through heterochronic parabiosis: insights into neurovascular coupling and the impact of young blood factors.

Age-related impairment of neurovascular coupling (NVC; "functional hyperemia") is a critical factor in the development of vascular cognitive impairment (VCI). Recent geroscience research indicates that cell-autonomous mechanisms alone cannot explain all aspects of neurovascular aging. Circulating factors derived from other organs, including pro-geronic factors (increased with age and detrimental to vascular homeostasis) and anti-geronic factors (preventing cellular aging phenotypes and declining with age), are thought to orchestrate cellular aging processes. This study aimed to investigate the influence of age-related changes in circulating factors on neurovascular aging. Heterochronic parabiosis was utilized to assess how exposure to young or old systemic environments could modulate neurovascular aging. Results demonstrated a significant decline in NVC responses in aged mice subjected to isochronic parabiosis (20-month-old C57BL/6 mice [A-(A)]; 6 weeks of parabiosis) when compared to young isochronic parabionts (6-month-old, [Y-(Y)]). However, exposure to young blood from parabionts significantly improved NVC in aged heterochronic parabionts [A-(Y)]. Conversely, young mice exposed to old blood from aged parabionts exhibited impaired NVC responses [Y-(A)]. In conclusion, even a brief exposure to a youthful humoral environment can mitigate neurovascular aging phenotypes, rejuvenating NVC responses. Conversely, short-term exposure to an aged humoral milieu in young mice accelerates the acquisition of neurovascular aging traits. These findings highlight the plasticity of neurovascular aging and suggest the presence of circulating anti-geronic factors capable of rejuvenating the aging cerebral microcirculation. Further research is needed to explore whether young blood factors can extend their rejuvenating effects to address other age-related cerebromicrovascular pathologies, such as blood-brain barrier integrity.

Pathological epigenetic events and reversibility review: the intersection between hallmarks of aging and developmental origin of health and disease.

We discuss pathological epigenetic events that are reversible (PEERs). A recent study by Poganik and colleagues showed that severe stress in mice and humans transiently elevates biological age of several tissues, and this transient age increase is reversible when the stress is removed. These studies suggest new strategies for reversing normal aging. However, it is important to note that developmental origin of health and disease studies have shown that developmental exposure to toxic chemicals such as lead causes permanent changes in neuron shape, connectivity and cellular hyperplasia of organs such as the heart and liver. In this review, the PEER hypothesis speculates that the hallmarks of aging and the hallmarks of developmental origin of health and disease intersect at PEERs.

The main goal in aging research is to find treatments to reverse aging. There are nine hallmarks of aging which describe cellular mechanisms that change as we age. For example, one of the hallmarks of aging is cellular senescence, which means that cells stop dividing when they get old. In this review, we describe nine hallmarks of developmental origins of health and disease (DOHaD). DOHaD studies show that exposures of the mother during pregnancy to stress or toxic chemicals can alter the health of the child throughout the child's lifespan. We argue that six of the nine hallmarks of DOHaD overlap with the hallmarks of aging and are reversible by dietary restriction or by drugs such as rapamycin which affect nutrient signaling. Based on this finding, we have formulated a hypothesis that we call 'pathological epigenetic events that are reversible' that contain the six hallmarks that overlap between the hallmarks of aging and the hallmarks of DOHaD. With this unexpected connection between aging and DOHaD, we argue that findings in one field, such as drugs that reverse aging, can apply to treatments in the other field, such as ways to reverse the adverse effects of exposures during pregnancy.

Remyelination and Ageing: Ethical Considerations of Using Surgically Joined Animals in Research.

Remyelination is a key repair process that ensures neurons remain protected following injury. This process is mediated by remyelinating oligodendrocytes in vertebrates, however, similarly to other neurobiological processes, the rate and efficiency of remyelination decreases across age and under pathological conditions. This has largely been attributed to two main contributors: 1) decreased exogenous signals supporting remyelination; and 2) aging of precursor cells that no longer differentiate into remyelinating oligodendrocytes. Here we discuss a key paper by Ruckh et al. (2012) who presented novel evidence that exposure to soluble bloodstream factors of young mice significantly rescues remyelination in old mice following a demyelinating insult. In this paper, a parabiosis approach was used where young and old mice were surgically joined for three weeks before and then left as a pair throughout the experiment. Ruckh and colleagues also offer novel insight into the role played by immune system cells, specifically macrophages, in clearance of myelin debris, a further contributor to remyelination. This paper is a good tool to expose undergraduate neuroscience students to basic molecular processes underlying conduction and transmission, helping them link cellular and network components. It also offers a platform for introducing the practicalities of in vivo research and debating ethical controversies that arise in animal research.

Heterochronic Plasma Transfer: Experimental Design, Considerations, and Technical Challenges.

Experimental approaches such as Heterochronic Plasma Transfer (HPT) provide insights into the aging process and help identify the factors that impact aging, with the aim of developing anti-aging therapies. HPT involves the transfer of plasma from an animal of one age to an animal of a different age and highlights the effects of the systemic environment on aging. Despite its importance as an aging research tool, HPT is not without limitations and HPT experiments across various studies differ in key experimental designs considerations, presenting a challenge in obtaining comparable outcomes. In this review, we examine the caveats and experimental design considerations of HPT as a research tool. We provide insights into plasma preparation procedures, route of administration, dosing regimen, and appropriate controls to assist investigators in achieving their experimental goals.

Secretome-based acellular therapy of bone marrow-derived mesenchymal stem cells in degenerative and immunological disorders: A narrative review.

The bone marrow (BM) plays a pivotal role in homeostasis by supporting hematopoiesis and immune cells' activation, maturation, interaction, and deployment. "BMSC-derived secretome" refers to the complete repertoire of secreted molecules, including nucleic acids, chemokines, growth factors, cytokines, and lipids from BM-derived mesenchymal stem cells (BMSCs). BMSC-derived secretomes are the current molecular platform for acellular therapy. Secretomes are highly manipulable and can be synthesised in vast quantities using commercially accessible cell lines in the laboratory. Secretomes are less likely to elicit an immunological response because they contain fewer surface proteins. Moreover, the delivery of BMSC-derived secretomes has been shown in numerous studies to be an effective, cell-free therapy method for alleviating the symptoms of inflammatory and degenerative diseases. As a result, secretome delivery from BMSCs has the same therapeutic effects as BMSCs transplantation but may have fewer adverse effects. Additionally, BMSCs' secretome has therapeutic promise for organoids and parabiosis studies. This review focuses on recent advances in secretome-based cell-free therapy, including its manipulation, isolation, characterisation, and delivery systems. The diverse bioactive molecules of secretomes that successfully treat inflammatory and degenerative diseases of the musculoskeletal, cardiovascular, nervous, respiratory, reproductive, gastrointestinal, and anti-ageing systems were also examined in this review. However, secretome-based therapy has some unfavourable side effects that may restrict its uses. Some of the adverse effects of this modal therapy were briefly mentioned in this review.

Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Programmed ageing: decline of stem cell renewal, immunosenescence, and Alzheimer's disease.

The characteristic maximum lifespan varies enormously across animal species from a few hours to hundreds of years. This argues that maximum lifespan, and the ageing process that itself dictates lifespan, are to a large extent genetically determined. Although controversial, this is supported by firm evidence that semelparous species display evolutionarily programmed ageing in response to reproductive and environmental cues. Parabiosis experiments reveal that ageing is orchestrated systemically through the circulation, accompanied by programmed changes in hormone levels across a lifetime. This implies that, like the circadian and circannual clocks, there is a master 'clock of age' (circavital clock) located in the limbic brain of mammals that modulates systemic changes in growth factor and hormone secretion over the lifespan, as well as systemic alterations in gene expression as revealed by genomic methylation analysis. Studies on accelerated ageing in mice, as well as human longevity genes, converge on evolutionarily conserved fibroblast growth factors (FGFs) and their receptors, including KLOTHO, as well as insulin-like growth factors (IGFs) and steroid hormones, as key players mediating the systemic effects of ageing. Age-related changes in these and multiple other factors are inferred to cause a progressive decline in tissue maintenance through failure of stem cell replenishment. This most severely affects the immune system, which requires constant renewal from bone marrow stem cells. Age-related immune decline increases risk of infection whereas lifespan can be extended in germfree animals. This and other evidence suggests that infection is the major cause of death in higher organisms. Immune decline is also associated with age-related diseases. Taking the example of Alzheimer's disease (AD), we assess the evidence that AD is caused by immunosenescence and infection. The signature protein of AD brain, Aß, is now known to be an antimicrobial peptide, and Aß deposits in AD brain may be a response to infection rather than a cause of disease. Because some cognitively normal elderly individuals show extensive neuropathology, we argue that the location of the pathology is crucial - specifically, lesions to limbic brain are likely to accentuate immunosenescence, and could thus underlie a vicious cycle of accelerated immune decline and microbial proliferation that culminates in AD. This general model may extend to other age-related diseases, and we propose a general paradigm of organismal senescence in which declining stem cell proliferation leads to programmed immunosenescence and mortality.

Understanding the role of the hematopoietic niche in Huntington's disease's phenotypic expression: in vivo evidence using a parabiosis model.

In a previous study, we have shown that parabiotic coupling of a knock-in mouse model (zQ175) of Huntington's disease (HD) to wild-type (WT) littermates resulted in a worsening of the normal phenotype as seen by detection of mutant huntingtin protein (mHTT) aggregates within peripheral organs and the cerebral cortex as well as vascular abnormalities in WT mice. In contrast, parabiosis improved disease features in the zQ175 mice such as reduction of mHTT aggregate number in the liver and cortex, decrease in blood-brain barrier (BBB) permeability and attenuation of mitochondrial impairments. While the shared circulation mediated these effects, no specific factor was identified. To better understand which blood elements were involved in the aforementioned changes, WT and zQ175 mice underwent parabiotic surgery prior to exposing one of the paired animals to irradiation. The irradiation procedure successfully eliminated the hematopoietic niche followed by repopulation with cells originating from the non-irradiated parabiont, as measured by the quantification of mHTT levels in peripheral blood mononuclear cells. Although irradiation of the WT parabiont, causing the loss of healthy hematopoietic cells, did lead to a few alterations in mitochondrial function in the muscle (TOM40 levels), and increased neuroinflammation in the striatum (GFAP levels), most of the changes observed were likely attributable to the irradiation procedure itself (e.g. mHTT aggregates in cortex and liver; cellular stress in peripheral organs). However, factors such as mHTT aggregation in the brain and periphery, and BBB leakage, which were improved in zQ175 mice when paired to WT littermates in the previous parabiosis experiment, were unaffected by perturbation of the hematopoietic niche. It would therefore appear that cells of the hematopoietic stem cell niche are largely uninvolved in the beneficial effects of parabiosis.

Heterochronic Parabiosis Causes Dacryoadenitis in Young Lacrimal Glands.

Aging is associated with inflammation and oxidative stress in the lacrimal gland (LG). We investigated if heterochronic parabiosis of mice could modulate age-related LG alterations. In both males and females, there were significant increases in total immune infiltration in isochronic aged LGs compared to that in isochronic young LGs. Male heterochronic young LGs were significantly more infiltrated compared to male isochronic young LGs. While both females and males had significant increases in inflammatory and B-cell-related transcripts in isochronic and heterochronic aged LGs compared to levels isochronic and heterochronic young LGs, females had a greater fold expression of some of these transcripts than males. Through flow cytometry, specific subsets of B cells were increased in the male heterochronic aged LGs compared to those in male isochronic aged LGs. Our results indicate that serum soluble factors from young mice were not enough to reverse inflammation and infiltrating immune cells in aged tissues and that there were specific sex-related differences in parabiosis treatment. This suggests that age-related changes in the LG microenvironment/architecture participate in perpetuating inflammation, which is not reversible by exposure to youthful systemic factors. In contrast, male young heterochronic LGs were significantly worse than their isochronic counterparts, suggesting that aged soluble factors can enhance inflammation in the young host. Therapies that aim at improving cellular health may have a stronger impact on improving inflammation and cellular inflammation in LGs than parabiosis.

Whole organism aging: Parabiosis, inflammaging, epigenetics, and peripheral and central aging clocks. The ARS of aging.

The strong interest shown in the study of the causes of aging in recent decades has uncovered many mechanisms that could contribute to the rate of aging. These include mitochondrial ROS production, DNA modification and repair, lipid peroxidation-induced membrane fatty acid unsaturation, autophagy, telomere shortening rate, apoptosis, proteostasis, senescent cells, and most likely there are many others waiting to be discovered. However, all these well-known mechanisms work only or mainly at the cellular level. Although it is known that organs within a single individual do not age at exactly the same rate, there is a well-defined species longevity. Therefore, loose coordination of aging rate among the different cells and tissues is needed to ensure species lifespan. In this article we focus on less known extracellular, systemic, and whole organism level mechanisms that could loosely coordinate aging of the whole individual to keep it within the margins of its species longevity. We discuss heterochronic parabiosis experiments, systemic factors distributed through the vascular system like DAMPs, mitochondrial DNA and its fragments, TF-like vascular proteins, and inflammaging, as well as epigenetic and proposed aging clocks situated at different levels of organization from individual cells to the brain. These interorgan systems can help to determine species longevity as a further adaptation to the ecosystem.

Bone-marrow derived cells do not contribute to new beta-cells in the inflamed pancreas.

The contribution of bone-marrow derived cells (BMCs) to a newly formed beta-cell population in adults is controversial. Previous studies have only used models of bone marrow transplantation from sex-mismatched donors (or other models of genetic labeling) into recipient animals that had undergone irradiation. This approach suffers from the significant shortcoming of the off-target effects of irradiation. Partial pancreatic duct ligation (PDL) is a mouse model of acute pancreatitis with a modest increase in beta-cell number. However, the possibility that recruited BMCs in the inflamed pancreas may convert into beta-cells has not been examined. Here, we used an irradiation-free model to track the fate of the BMCs from the donor mice. A ROSA-mTmG red fluorescent mouse was surgically joined to an INS1Cre knock-in mouse by parabiosis to establish a mixed circulation. PDL was then performed in the INS1Cre mice 2 weeks after parabiosis, which was one week after establishment of the stable blood chimera. The contribution of red cells from ROSA-mTmG mice to beta-cells in INS1Cre mouse was evaluated based on red fluorescence, while cell fusion was evaluated by the presence of green fluorescence in beta-cells. We did not detect any red or green insulin+ cells in the INS1Cre mice, suggesting that there was no contribution of BMCs to the newly formed beta-cells, either by direct differentiation, or by cell fusion. Thus, the contribution of BMCs to beta-cells in the inflamed pancreas should be minimal, if any.